Azacytosine analogs and derivatives

ABSTRACT

Compounds and compositions of azacytosine analogs and derivatives are provided. In one aspect of the invention, analogs or derivatives of decitabine and azacitidine are provided with modification at the 2-, 4-, or 6-position of the triazine ring, at the 1′-6′position of the ribose ring, or combinations thereof. Methods of using, synthesizing and manufacturing these analogs and derivatives are also provided. These compounds can be formulated into pharmaceutical compositions that can be used for treating any disease associated with aberrant DNA methylation, or a disease or condition that is sensitive to the treatment with decitabine or azacitidine, such as hematological disorders, tumors and cancers.

CROSS-REFERENCE

This application is a continuation-in-part application of Ser. No.11/077,862, filed Mar. 11, 2005, which is incorporated herein byreference in its entirety and to which application we claim priorityunder 35 USC §120.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds and compositions of cytosineanalogs and derivatives, and methods for preparing, formulating andadministering these compounds or compositions to a host in need thereof.

2. Description of Related Art

A few nucleosides of azacytosine, such as 5-aza-2′-deoxycytidine (alsocalled decitabine, FIG. 1A, structure 1a) and azacitidine (FIG. 1A,structure 1b), have been developed as antagonist of its related naturalnucleoside, cytidine and 2′-deoxycytidine, respectively. The onlystructural difference between azacytosine and cytosine is the presenceof a nitrogen atom at position 5 of the cytosine ring in azacytosine ascompared to a carbon at this position for cytosine. Decitabine (1a) wasprepared by cyclization of peracylglycosyl isocyanates (Pliml and Sorm(1964) Collect. Czech. Chem. Commun. 29:2576-2577); later azacitidine(1b) and a series of related analogues were prepared (Naeem et. al.(1998) Collect. Czech. Chem. Commun. 63:222-230).

Two isomeric forms of decitabine can be distinguished. The β-anomer isthe active form. The modes of decomposition of decitabine in aqueoussolution are (a) conversion of the active β-anomer to the inactiveα-anomer (Pompon et al. (1987) J. Chromat. 388:113-122); (b) ringcleavage of the aza-pyrimidine ring to formN—(formylamidino)-N′-β-D-2′-deoxy-(ribofuranosy)-urea (Mojaverian andRepta (1984) J. Pharm. Pharmacol. 36:728-733); and (c) subsequentforming of guanidine compounds (Kissinger and Stemm (1986) J. Chromat.353:309-318).

Decitabine possesses multiple pharmacological characteristics. At amolecular level, it is S-phase dependent for incorporation into DNA. Ata cellular level, decitabine can induce cell differentiation and exerthematological toxicity. Despite having a short half life in vivo,decitabine has an excellent tissue distribution.

One of the function of decitabine is its ability to specifically andpotently inhibit DNA methylation. Methylation of cytosine to5-methylcytosine occurs at the level of DNA. Inside the cell, decitabineis first converted into its active form, the phosphorylated5-aza-deoxycytidine, by deoxycytidine kinase which is primarilysynthesized during the S phase of the cell cycle. The affinity ofdecitabine for the catalytical site of deoxycytidine kinase is similarto the natural substrate, deoxycytidine. Momparler et al. (1985)30:287-299. After conversion to its triphosphate form by deoxycytidinekinase, decitabine is incorporated into replicating DNA at a ratesimilar to that of the natural substrate, dCTP. Bouchard and Momparler(1983) Mol. Pharmacol. 24:109-114.

Incorporation of decitabine into the DNA strand has a hypomethylationeffect. Each class of differentiated cells has its own distinctmethylation pattern. After chromosomal duplication, in order to conservethis pattern of methylation, the 5-methylcytosine on the parental strandserves to direct methylation on the complementary daughter DNA strand.Substituting the carbon at the 5 position of the cytosine for a nitrogeninterferes with this normal process of DNA methylation. The replacementof 5-methylcytosine with decitabine at a specific site of methylationproduces an irreversible inactivation of DNA methyltransferase,presumably due to formation of a covalent bond between the enzyme anddecitabine. Juttermann et al. (1994) Proc. Natl. Acad. Sci. USA91:11797-11801. By specifically inhibiting DNA methyltransferase, theenzyme required for methylation, the aberrant methylation of the tumorsuppressor genes can be prevented.

Decitabine is commonly supplied as a sterile lyophilized powder forinjection, together with buffering salt, such as potassium dihydrogenphosphate, and pH modifier, such as sodium hydroxide. For example,decitabine is supplied by SuperGen, Inc., as lyophilized powder packedin 20 mL glass vials, containing 50 mg of decitabine, monobasicpotassium dihydrogen phosphate, and sodium hydroxide. When reconstitutedwith 10 mL of sterile water for injection, each mL contain 5 mg ofdecitabine, 6.8 mg of KH₂PO₄, and approximately 1.1 mg NaOH. The pH ofthe resulting solution is 6.5-7.5. The reconstituted solution can befurther diluted to a concentration of 1.0 or 0.1 mg/mL in cold infusionfluids, i.e., 0.9% Sodium Chloride; or 5% Dextrose; or 5% Glucose; orLactated Ringer's. The unopened vials are typically stored underrefrigeration (2-8° C.; 36-46° F.), in the original package.

Decitabine is most typically administrated to patients by injection,such as by a bolus I.V. injection, continuous I.V. infusion, or I.V.infusion. The length of I.V. infusion is limited by the decomposition ofdecitabine in aqueous solutions.

Thus, a need still exists for compounds, compositions and methods forimproving chemical stability of cytosine analogs, especially in aqueoussolutions. The present invention provides such improvements.

SUMMARY OF THE INVENTION

Compounds, salts and compositions of cytosine analogs and derivativesare provided. In one aspect of the invention, analogs or derivatives ofdecitabine and azacitidine are provided with modification at the 2-, 4-,or 6-position of the triazine ring, at the 1 ′-6′position of the ribosering, or combinations thereof.

In one embodiment, a compound of Formula I is provided.

In a variant of the embodiment, group R_(x) of Formula I is a halogen(i.e., fluoride, chloride, bromide, or iodide), and preferably fluoride.

According to the embodiment, group R_(y) of Formula I is preferably ahydrogen, alkyl, aryl, or sugar, and more preferably a substituted orunsubstituted ribofuranose or 2′-deoxyribofuranose.

According to the embodiment, group R_(z) of Formula I is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, or hydroxylalkylamino.

According to the embodiment, group Q of Formula I is oxygen, sulfur,methylene, imine, or alkylimine.

In another variant of the embodiment, group R_(x) of Formula I ispreferably a strong electron-donating group such as hydroxyl, thiol,primary, secondary or tertiary amino, —O-alkyl, —S-alkyl, —O-aryl and—S-aryl. Strong electron-donating group is defined as the class ofchemical moieties that can decrease the electrophilicity of an adjacentsite by resonance or inductive effects.

In yet another variant of the embodiment, group R_(x) of Formula I ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) ishydrogen, methyl, chloromethyl, phenyl or benzyl, Q is oxygen and R_(y)is hydrogen or glycosyl, R_(z) is not amino.

In yet another variant of the embodiment, group R_(x) of Formula I ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) is alkylhaving 1 to 4 carbon atoms, Q is oxygen and R_(y) is hydrogen orglycosyl, R_(z) is not amino, alkylamino, or dialkylamino with the alkylhaving 1-4 carbon atoms, or aralkylamino or diaralkylamino with thearalkyl having 7 to 10 carbon atoms.

In another embodiment, a compound of Formula II is provided.

In a variant of the embodiment, group R_(x) of Formula II is a halogen(i.e., fluoride, chloride, bromide, or iodide), and preferably fluoride.

In another variant of the embodiment, group R_(x) of Formula II ispreferably a strong electron-donating group such as hydroxyl, thiol,primary, secondary or tertiary amino, —O-alkyl, —S-alkyl, —O-aryl and—S-aryl.

According to the embodiment, group R_(z) of Formula II is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, and hydroxylalkylamino.

According to the embodiment, group Q of Formula II is oxygen, sulfur,methylene, imine, or alkylimine.

In yet another variant of the embodiment, group R_(x) of Formula II ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) ishydrogen, methyl, chloromethyl, phenyl or benzyl, Q is oxygen and R_(y)is hydrogen or glycosyl, R_(z) is not amino.

In yet another variant of the embodiment, group R_(x) of Formula II ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) is alkylhaving 1 to 4 carbon atoms, Q is oxygen and R_(y) is hydrogen orglycosyl, R_(z) is not amino, alkylamino, or dialkylamino with the alkylhaving 1-4 carbon atoms, or aralkylamino or diaralkylamino with thearalkyl having 7 to 10 carbon atoms.

In yet another embodiment, a compound of Formula III is provided.

In a variant of the embodiment, group R_(x) of Formula III is a halogen(i.e., fluoride, chloride, bromide, or iodide), and preferably fluoride.

In another variant of the embodiment, group R_(x) of Formula III ispreferably a strong electron-donating group such as hydroxyl, thiol,amino, —N-alkyl, —O-alkyl, —S-alkyl, —N-aryl, —O-aryl and —S-aryl;

According to the embodiment, group R_(z) of Formula III is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, and hydroxylalkylamino.

According to the embodiment, group Q of Formula III is oxygen, sulfur,methylene, imine, or alkylimine.

In yet another variant of the embodiment, group R_(x) of Formula III ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) ishydrogen, methyl, chloromethyl, phenyl or benzyl, Q is oxygen and R_(y)is hydrogen or glycosyl, R_(z) is not amino.

In yet another variant of the embodiment, group R_(x) of Formula III ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) is alkylhaving 1 to 4 carbon atoms, Q is oxygen and R_(y) is hydrogen orglycosyl, R_(z) is not amino, alkylamino, or dialkylamino with the alkylhaving 1-4 carbon atoms, or aralkylamino or diaralkylamino with thearalkyl having 7 to 10 carbon atoms.

In yet another embodiment, a compound of Formula IV is provided.

In a variant of the embodiment, group R_(x) of Formula IV is hydrogen,alkyl (preferably straight or branched chain C₁₋₆ alkyl), aryl,halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl.

In another variant of the embodiment, group R_(x) of Formula IV is ahalogen (i.e., fluoride, chloride, bromide, or iodide), and preferablyfluoride.

In yet another variant of the embodiment, group R_(x) of Formula IV ispreferably an electron-donating group, and more preferably a strongelectron-donating group such as hydroxyl, thiol, amino, —N-alkyl,—O-alkyl, —S-alkyl, —N-aryl, —O-aryl and —S-aryl.

According to the embodiment, group R_(z) of Formula IV is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, and hydroxylalkylamino.

According to the embodiment, group Q of Formula IV is oxygen, sulfur,methylene, imine, or alkylimine.

According to the embodiment, group R₁ ^(′), R₂ ^(′), R₃ ^(′), R₄ ^(′),R₅ ^(′), or R₆ ^(′) of Formula IV is each independently selected fromthe group consisting of hydrogen, hydroxyl, fluoride, choloride,bromide, iodide, CF₃, —O-alkyl, —O-acyl, —O-aryl, —S-alkyl, and —S-aryl,provided that when R_(x) is hydrogen and R_(z) is amino, R₄ ^(′) is nothydroxyl. Preferably, R₄ ^(′) is hydrogen and R₁ ^(′), R₂ ^(′), R₃ ^(′),R₅ ^(′), or R₆ ^(′) is independently hydrogen, fluoride, chloride,bromide, iodide, CF₃, —O-alkyl, —O-acyl, —O-aryl, —S-alkyl, or —S-aryl.

The invention also provides a salt form of azacytosine analogs andderivatives, more preferably pharmaceutically-acceptable salts of thecompounds of the invention (preferably a compound of Formulas I-IV).

In one embodiment, salt of a compound of Formula I is provided,

wherein R_(x) is hydrogen, alkyl, aryl, halogen-substituted alkyl oraryl, phenyl, or benzyl; R_(y) is hydrogen, alkyl, or sugar; R_(z) is ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, or hydroxylalkylamino; and Q is oxygen,sulfur, methylene, imine, or alkylimine, provided that when R_(y) is2′-deoxy-D-ribose or D-ribose, R_(x) is not hydrogen, and R_(z) is notamino.

According to the embodiment, the salt may be synthesized with an acidsuch as hydrochloric, L-lactic, acetic, phosphoric, (+)-L-tartaric,citric, propionic, butyric, hexanoic, L-aspartic, L-glutamic, succinic,EDTA, maleic, and methanesulfonic acid; HBr, HF, HI, nitric, nitrous,sulfuric, sulfurous, phosphorous, perchloric, chloric, and chlorousacid; carboxylic acid such as ascorbic, carbonic, and fumaric acid;sulfonic acid such as ethanesulfonic, 2-hydroxyethanesulfonic, andtoluenesulfonic acid;.

Also according to the embodiment, the salt is preferably ahydrochloride, mesylate, EDTA, sulfite, L-Aspartate, maleate, phosphate,L-Glutamate, (+)-L-Tartrate, citrate, L-Lactate, succinate, acetate,hexanoate, butyrate, or propionate salt.

Methods of synthesizing and manufacturing these analogs and derivativesare also provided. These compounds can be formulated into pharmaceuticalcompositions that can be used for treating any disease that is sensitiveto the treatment with decitabine or azacitidine, such as hematologicaldisorders and cancer.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates chemical structures of decitabine and azacitidine.

FIG. 2 illustrates chemical structures of cytarabine and 6-azacytidine.

FIG. 3A is a scheme of hydrolysis of decitabine in acidic medium.

FIG. 3B is a scheme of hydrolysis of decitabine in neutral medium.

FIG. 3C is a schematic illustration of resonance of the triazine ring ofdecitabine and azacitidine.

FIGS. 3D is a scheme of deamination of decitabine by cytidine deaminase.

FIGS. 4A illustrates the strategy of modifying the 4- and 6-position ofthe triazine ring of decitabine in order to improve its chemical andphysiological stability.

FIGS. 4B-D illustrate examples of decitabine analogs with modificationsin the 4- and/or 6-position of the triazine ring, as well as in the2′-deoxyribose ring.

FIG. 4E illustrates schemes for synthesis of various analogs orderivatives of decitabine and azacitidine.

FIG. 4F illustrates a scheme for modifying the 4-position of an analogof decitabine or azacitidine.

FIG. 5A illustrates a scheme for modification of the N═CH functionalgroup of the triazine ring of decitabine or azacitidine.

FIG. 5B illustrates a scheme for synthesis of keto-enol derivatives ofdecitabine or azacitidine.

FIG. 6A illustrates a scheme for modifying the 1′-6′-position ofdecitabine or azacitidine.

FIGS. 6B-C illustrate examples of decitabine or azacitidine analogs withmodifications in the 4- and/or 6-position of the triazine ring, as wellas in the deoxyribose or ribose ring.

FIG. 6D illustrates electron resonance analysis of the triazine ringwith modifications in the 6-position.

FIG. 6E illustrates electron resonance analysis of the triazine ringwith modifications in the 4-position.

FIG. 7A illustrates a scheme for synthesis of halogen derivatives ofdecitabine by using halogenated pentose and triazine precursors.

FIG. 7B illustrates a scheme for synthesis of halogen derivatives ofazacitidine.

FIG. 7C illustrates a synthetic scheme for halogenation of the triazinering of decitabine or azacitidine.

FIG. 7D illustrates a scheme for synthesis of 6-fluoride derivatives ofdecitabine or azacitidine.

FIGS. 8A-B illustrate examples of decitabine and azacitidine derivativeswith combined modifications at the 6-position and at the 2′, 3′ and/or5′ position.

FIG. 8C illustrates a scheme for synthesis of an analogousfluoro-intermediate.

FIGS. 8D-G illustrate examples of decitabine and azacitidine derivativeswith combined modifications at the 4-position and at the 2′, 3′ and/or5′ position.

FIG. 8H illustrates a scheme for synthesis of an analogousfluoro-intermediate.

FIG. 9 illustrates a scheme for synthesis of compound 1′d.

FIG. 10 illustrates a scheme for synthesis of compound 1′a.

FIG. 11 illustrates a scheme for synthesis of compound 7a.

FIG. 12 illustrates a scheme for synthesis of compound 134.

FIG. 13 illustrates a scheme for synthesis of compound 7c.

FIG. 14 illustrates a scheme for synthesis of compound 135.

FIG. 15 illustrates a scheme for synthesis of compound 136.

FIG. 16 illustrates a scheme for synthesis of compound 137.

FIG. 17 is a pH-solubility and pH-degradation profile of decitabine inone hour.

FIG. 18 lists various analogs and derivatives of decitabine.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The present invention provides novel azacytosine analogs andderivatives, pharmaceutically acceptable salts and composition thereof.The invention circumvents limitations of DNA methylation inhibitors suchas decitabine and azacitidine, including fast decomposition in aqueoussolutions and discomfort to patients due to cold infusions of decitabineand azacitidine. Leveraging their knowledge based on analysis of studiesconducted on hydrolysis of decitabine and azacitidine under variousconditions and analysis of resonance of the triazine ring of these twocompounds, the inventors provide solutions to the instability problemsof azacytidine compounds through modification of the triazine ring andthe ribose ring. Compared to the unmodified azacytidines, the inventivecompounds and compositions should have superior long-term chemicalstability, convenient storage and administration, and cause lessdiscomfort to patients due to cold infusions.

The compounds and compositions of present invention can be used to treatpatient suffering from a disease sensitive to the treatment withdecitabine or azacitidine, such as hematological disorders, benigntumors, malignant tumors, restenosis, and inflammatory diseases viavarious routes of administration such as intravenous, intramuscular,subcutaneous injection, oral administration and inhalation.

The present invention also provides methods for designing chemicallystable azacytosine analogs, methods of synthesizing, formulating andmanufacturing these compounds and compositions thereof.

The following is a detailed description of the invention and preferredembodiments of the inventive compounds, compositions, methods of use,synthesis, formulation and manufacture.

1. Problems of Chemical and Physiological Instability of Decitabine andAzacitidine and Solutions Provided by the Invention

Decitabine and azacitidine are unstable in aqueous media and undergohydrolytic degradation. In acidic medium, decitabine is hydrolyzed atroom temperature to 5-azacytosine (1e) and 2-deoxyribose (1f, FIG. 3A).In neutral medium at room temperature, the opening of the triazine ringtakes place at the 6-position to form the transient intermediate formylderivative (1g), which is further hydrolyzed to the amidino-ureaderivative (1h) and formic acid (FIG. 3B) (Piskala, A.; Synackova, M.;Tomankova, H.; Fiedler, P.; Zizkowsky, V. Nucleic Acids Res. 1978, 4,s109-s-113.). This hydrolysis at the 6-position also occurs in acidicand basic aqueous media at even faster rates.

The inventors believe that the hydrolysis is due to the fact that the6-position is very electrophilic and open to nucleophilic attack by awater molecule, which leads to hydrolytic cleavage. Based on thistheory, resonance analysis of the triazine ring was conducted to showthat the 6-position is both electrophilic and less sterically hinderedthan the next electrophilic site (FIG. 3C): the 6-position carbocation(secondary carbon) is bonded with only two nitrogen atoms, while the4-position carbocation tertiary carbon) is bonded with three nitrogenatoms. This supports the inventor's belief and explains why the6-position of the triazine ring is liable to undergo hydrolyticcleavage.

As described above, in aqueous media of varying pH, decitabine undergoesrapid hydrolytic cleavages. In alkaline medium the hydrolysis to theamidino-urea derivative (1h) occurs even faster, within a few minutes.Azacitidine also undergoes similar degradation in aqueous media at allpHs.

Decitabine and azacitidine are also unstable in biological fluid due todeamination. The deamination of decitabine to 5-aza-2′-deoxyuridine iscatalyzed by cytidine deaminase. Chabot et al. (1983) BiochemicalPharmacology 22:1327-1328. The estimated K_(m) of decitabine was 250 μMfor the enzyme purified from human liver as compared to the K_(m) of 12μM for the natural substrate, deoxycytidine. The rate of deamination ofdeoxycytidine was about 6-fold greater than that of decitabine bycytidine deaminase at equal concentrations.

The enol derivative after deamination (1i, FIG. 3D) should have retainedits capacity to bind and inhibit methyltransferase; however, the morethermodynamically favorable keto tautomer (1i′) has a carbonylfunctional group at the 4-position, which makes the 6-position highlyelectrophilic and undergoes hydrolytic cleavage. Thus, a possiblemechanism of resistance to decitabine is an increase in the levels ofthe deaminating enzyme cytidine deaminase. Treatment with decitabine hasbeen associated with an increase in the cytidine deaminase activity inHL-60 cells and in leukemic cells in some patients. To increase thestability of cytosine nucleotides such as decitabine and azacitidine,the inventors believe that this susceptibility toward deamination andpredisposition toward hydrolytic cleavage of the triazine ring beforeand after deamination of the 4-position NH2 group by cytidine deaminaseshould be removed by modifying the ring in various ways as providedbelow (examples shown in FIG. 4B, C, D; 6B, C; 8D, E, F, G). When the4-position is substituted by stronger bases (1′b-1′g, 1″b-1″g, FIG. 4D)or substituted by an equivalent electron-donating group that does nottautomerize (1′a, 1″a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, FIG.4B, C, D), the potential for deamination and subsequent hydrolyticcleavage is minimized. When the 6-position is substitutedelectron-donating groups (FIG. 4B, C; 6B, C; 8A, B), the propensitytoward hydrolytic cleavage is also minimized.

The inventors believe that the inventive compounds represent a newgeneration of hypomethylating agents that not only retain the uniquemechanism of action of decitabine and azacitidine but also have improvedaqueous stability and activity. Increased stability of the inventivecompounds should make manufacturing of the active pharmaceuticalingredient (API) and drug product more robust and economical, facilitatedevelopment of different, more patient-friendly formulations, andincrease bioavailability of the drug.

Preferred embodiments of the inventive compounds are described in detailin the following section.

2. Azacytosine Analogs and Derivatives According to the Invention

The present invention provides azacytosine analogs and derivatives withimproved chemical stability in aqueous solution and against cytidinedeaminase. Preferred embodiments are shown as chemical formula ordescribed in the text of the specification.

As used herein, the term “alkyl” refers to a saturated or unsaturated,branched, straight-chain or cyclic monovalent hydrocarbon group derivedby the removal of one hydrogen atom from a single carbon atom of aparent alkane, alkene or alkyne. Typical alkyl groups include, but arenot limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl;propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl),cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

In addition, the term “alkyl” is specifically intended to include groupshaving any degree or level of saturation, i.e., groups havingexclusively single carbon-carbon bonds, groups having one or more doublecarbon-carbon bonds, groups having one or more triple carbon-carbonbonds and groups having mixtures of single, double and triplecarbon-carbon bonds. Where a specific level of saturation is intended,the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used.Preferably, an alkyl group comprises from 1 to 20 carbon atoms, morepreferably, from 1 to 10 carbon atoms, and most preferably from 1-5carbon atoms.

As used herein, the term “alkanyl” refers to a saturated branched,straight-chain or cyclic alkyl group derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane. Typicalalkanyl groups include, but are not limited to, methanyl; ethanyl;propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl,etc.; butanyls such as butan-1-yl, butan-2-yl(sec-butyl),2-methyl-propan-1-yl(isobutyl), 2-methyl-propan-2-yl(t-butyl),cyclobutan-1-yl, etc.; and the like.

As used herein, the term “alkenyl” refers to an unsaturated branched,straight-chain or cyclic alkyl group having at least one carbon-carbondouble bond derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkene. The group may be in either the cis ortrans conformation about the double bond(s). Typical alkenyl groupsinclude, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like.

As used herein, the term “alkynyl” refers to an unsaturated branched,straight-chain or cyclic alkyl group having at least one carbon-carbontriple bond derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkyne. Typical alkynyl groups include, but arenot limited to, ethynyl; propynyls such as prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl,but-3-yn-1-yl, etc.; and the like.

As used herein, the term “acyl” refers to a group —C(O)R, where R ishydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.Representative examples include, but are not limited to formyl, acetyl,cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyland the like.

As used herein, the term “acylamino” (or alternatively “acylamido”)refers to a group —NR′C(O)R, where R′ and R are each independentlyhydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein.Representative examples include, but are not limited to, formylamino,acetylamino (i.e., acetamido), cyclohexylcarbonylamino,cyclohexylmethylcarbonylamino, benzoylamino (i.e., benzamido),benzylcarbonylamino and the like.

As used herein, the term “acyloxy” refers to a group —OC(O)R, where R ishydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroalkyl, heteroaryl or heteroarylalkyl, as defined herein.Representative examples include, but are not limited to, acetyloxy (oracetoxy), butyloxy (or butoxy), benzoyloxy and the like.

As used herein, the term “alkylamino” means a group —NHR where Rrepresents an alkyl or cycloalkyl group as defined herein.Representative examples include, but are not limited to, methylamino,ethylamino, 1-methylethylamino, cyclohexyl amino and the like.

As used herein, the term “alkoxy” refers to a group —OR where Rrepresents an alkyl or cycloalkyl group as defined herein.Representative examples include, but are not limited to, methoxy,ethoxy, propoxy, butoxy, cyclohexyloxy and the like.

As used herein, the term “alkoxycarbonyl” refers to a group —C(O)-alkoxywhere alkoxy is as defined herein.

As used herein, the term “alkylsulfonyl” refers to a group —S(O)₂R whereR is an alkyl or cycloalkyl group as defined herein. Representativeexamples include, but are not limited to, methylsulfonyl, ethylsulfonyl,propylsulfonyl, butylsulfonyl and the like.

As used herein, the term “alkylsulfinyl” refers to a group —S(O)R whereR is an alkyl or cycloalkyl group as defined herein. Representativeexamples include, but are not limited to, methylsulfinyl, ethylsulfinyl,propylsulfinyl, butylsulfinyl and the like.

As used herein, the term “alkylthio” refers to a group —SR where R is analkyl or cycloalkyl group as defined herein that may be optionallysubstituted as defined herein. Representative examples include, but arenot limited to methylthio, ethylthio, propylthio, butylthio and thelike.

As used herein, the term “amino” refers to the group —NH₂.

As used herein, the term “aryl” refers to a monovalent aromatichydrocarbon group derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, groups derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexalene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthaleneand the like. Preferably, an aryl group comprises from 6 to 20 carbonatoms, more preferably between 6 to 12 carbon atoms.

As used herein, the term “arylalkyl” refers to an acyclic alkyl group inwhich one of the hydrogen atoms bonded to a carbon atom, typically aterminal or sp³ carbon atom, is replaced with an aryl group. Typicalarylalkyl groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. Preferably, an arylalkyl group is (C₆-C₃₀)arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkylgroup is (C₁-C₁₀) and the aryl moiety is (C₆-C₂₀), more preferably, anarylalkyl group is (C₆-C₂₀) arylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the arylalkyl group is (C₁-C₈) and the aryl moiety is(C₆-C₁₂).

As used herein, the term “arylalkyloxy” refers to an —O-arylalkyl groupwhere arylalkyl is as defined herein.

As used herein, the term “aryloxycarbonyl” refers to a group—C(O)—O-aryl where aryl is as defined herein.

As used herein, the terms “hydroxylalkylamino” and “hydroxylarylamino”refer to a group —NHROH where R represents an alkyl or aryl substitutedwith a hydroxyl —OH group.

As used herein, the term “sugar” refers to saccharides (Greek:sakcharon, sugar) or carbohydrates, which includes monosaccharides,oligosaccharides, and polysaccharides. In particular, ribose and2′-deoxyribose belong to the subgroup of pentose monosaccharides.

As used herein, the term “glycosyl” refers to a structure obtained byremoving the hydroxy group from the hemiacetal function of amonosaccharide and, by extension, of a lower oligosaccharide. See IUPACCompendium of Chemical Terminology (1995) 67:1338.

As used herein, the terms “thiolalkylamino” and “thiolarylamino” referto a group —NHRSH where R represents an alkyl or aryl substituted with athiol —SH group.

In one embodiment, a compound of Formula I is provided.

In a variant of the embodiment, group R_(x) of Formula I is a halogen(i.e., fluoride, chloride, bromide, or iodide), and preferably fluoride.

According to the embodiment, group R_(y) of Formula I is preferably ahydrogen, alkyl, aryl, or sugar, and more preferably a substituted orunsubstituted ribofuranose or 2′-deoxyribofuranose.

According to the embodiment, group R_(z) of Formula I is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, or hydroxylalkylamino.

According to the embodiment, group Q of Formula I is oxygen, sulfur,methylene, imine, or alkylimine.

In another variant of the embodiment, group R_(x) of Formula I ispreferably a strong electron-donating group such as hydroxyl, thiol,primary, secondary or tertiary amino, —O-alkyl, —S-alkyl, —O-aryl and—S-aryl. Strong electron-donating group is defined as the class ofchemical moieties that can decrease the electrophilicity of an adjacentsite by resonance or inductive effects.

In yet another variant of the embodiment, group R_(x) of Formula I ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) ishydrogen, methyl, chloromethyl, phenyl or benzyl, Q is oxygen and R_(y)is hydrogen or glycosyl, R_(z) is not amino.

In yet another variant of the embodiment, group R_(x) of Formula I ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) is alkylhaving 1 to 4 carbon atoms, Q is oxygen and R_(y) is hydrogen orglycosyl, R_(z) is not amino, alkylamino, or dialkylamino with the alkylhaving 1-4 carbon atoms, or aralkylamino or diaralkylamino with thearalkyl having 7 to 10 carbon atoms.

In another embodiment, a compound of Formula II is provided.

In a variant of the embodiment, group R_(x) of Formula II is a halogen(i.e., fluoride, chloride, bromide, or iodide), and preferably fluoride.

In another variant of the embodiment, group R_(x) of Formula II ispreferably a strong electron-donating group such as hydroxyl, thiol,primary, secondary or tertiary amino, —O-alkyl, —S-alkyl, —O-aryl and—S-aryl.

According to the embodiment, group R_(z) of Formula II is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, and hydroxylalkylamino.

According to the embodiment, group Q of Formula II is oxygen, sulfur,methylene, imine, or alkylimine.

In yet another variant of the embodiment, group R_(x) of Formula II ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) ishydrogen, methyl, chloromethyl, phenyl or benzyl, Q is oxygen and R_(y)is hydrogen or glycosyl, R_(z) is not amino.

In yet another variant of the embodiment, group R_(x) of Formula II ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) is alkylhaving 1 to 4 carbon atoms, Q is oxygen and R_(y) is hydrogen orglycosyl, R_(z) is not amino, alkylamino, or dialkylamino with the alkylhaving 1-4 carbon atoms, or aralkylamino or diaralkylamino with thearalkyl having 7 to 10 carbon atoms.

In yet another embodiment, a compound of Formula III is provided.

In a variant of the embodiment, group R_(x) of Formula III is a halogen(i.e., fluoride, chloride, bromide, or iodide), and preferably fluoride.

In another variant of the embodiment, group R_(x) of Formula III ispreferably a strong electron-donating group such as hydroxyl, thiol,amino, —N-alkyl, —O-alkyl, —S-alkyl, —N-aryl, —O-aryl and —S-aryl.

According to the embodiment, group R_(z) of Formula III is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, and hydroxylalkylamino.

According to the embodiment, group Q of Formula III is oxygen, sulfur,methylene, imine, or alkylimine.

In yet another variant of the embodiment, group R_(x) of Formula III ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) ishydrogen, methyl, chloromethyl, phenyl or benzyl, Q is oxygen and R_(y)is hydrogen or glycosyl, R_(z) is not amino.

In yet another variant of the embodiment, group R_(x) of Formula III ishydrogen, alkyl (preferably straight or branched chain C₁₋₆ alkyl),aryl, halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl, provided that when R_(x) is alkylhaving 1 to 4 carbon atoms, Q is oxygen and R_(y) is hydrogen orglycosyl, R_(z) is not amino, alkylamino, or dialkylamino with the alkylhaving 1-4 carbon atoms, or aralkylamino or diaralkylamino with thearalkyl having 7 to 10 carbon atoms.

In yet another embodiment, a compound of Formula IV is provided.

In a variant of the embodiment, group R_(x) of Formula IV is hydrogen,alkyl (preferably straight or branched chain C₁₋₆ alkyl), aryl,halogen-substituted alkyl or aryl (preferably mono-, di- ortrifluoromethyl), phenyl, or benzyl.

In another variant of the embodiment, group R_(x) of Formula IV is ahalogen (i.e., fluoride, chloride, bromide, or iodide), and preferablyfluoride.

In yet another variant of the embodiment, group R_(x) of Formula IV ispreferably an electron-donating group, and more preferably a strongelectron-donating group such as hydroxyl, thiol, amino, —N-alkyl,—O-alkyl, —S-alkyl, —N-aryl, —O-aryl and —S-aryl.

According to the embodiment, group R_(z) of Formula IV is preferably ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, and hydroxylalkylamino.

According to the embodiment, group Q of Formula IV is oxygen, sulfur,methylene, imine, or alkylimine.

According to the embodiment, group R₁ ^(′), R₂ ^(′), R₃ ^(′), R₄ ^(′),R₅ ^(′), or R₆ ^(′) of Formula IV is each independently selected fromthe group consisting of hydrogen, hydroxyl, fluoride, choloride,bromide, iodide, CF₃, —O-alkyl, —O-acyl, —O-aryl, —S-alkyl, and —S-aryl,provided that when R_(x) is hydrogen and R_(z) is amino, R₄ ^(′) is nothydroxyl. Preferably, R₄ ^(′) is hydrogen and R₁ ^(′), R₂ ^(′), R₃ ^(′),R₅ ^(′), or R₆ ^(′) is independently hydrogen, fluoride, chloride,bromide, iodide, CF₃, —O-alkyl, —O-acyl, —O-aryl, —S-alkyl, or —S-aryl.

The compounds of the invention (preferably compounds of Formulas I-IV)can possess one or more asymmetric carbon atoms and are thus capable ofexisting in the form of optical isomers as well as in the form ofracemic or non-racemic mixtures thereof. The compounds of the invention(preferably compounds of Formulas I-IV) can be utilized in the presentinvention as a single isomer or as a mixture of stereochemical isomericforms.

Diastereoisomers can be separated by conventional means such aschromatography, distillation, crystallization or sublimation. Theoptical isomers can be obtained by resolution of the racemic mixturesaccording to conventional processes, for example by formation ofdiastereoisomeric salts by treatment with an optically active acid orbase. Examples of appropriate acids are tartaric, diacetyltartaric,dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. Themixture of diastereomers can be separated by crystallization followed byliberation of the optically active bases from these salts.

An alternative process for separation of optical isomers includes theuse of a chiral chromatography column optimally chosen to maximize theseparation of the enantiomers. Still another available method involvessynthesis of covalent diastereoisomeric molecules by reacting compoundsof the invention (preferably compounds of Formula I) with an opticallypure acid in an activated form or an optically pure isocyanate. Thesynthesized diastereoisomers can be separated by conventional means suchas chromatography, distillation, crystallization or sublimation, andthen hydrolyzed to obtain the enantiomerically pure compound. Theoptically active compounds of the invention (preferably compounds ofFormulas I-IV) can likewise be obtained by utilizing optically activestarting materials. These isomers may be in the form of a free acid, afree base, an ester or a salt.

The invention also embraces pharmaceutically-acceptable salts of thecompounds of the invention (preferably a compound of Formulas I-IV).

In one embodiment, salt of a compound of Formula I is provided,

wherein R_(x) is hydrogen, alkyl, aryl, halogen-substituted alkyl oraryl, phenyl, or benzyl; R_(y) is hydrogen, alkyl, or sugar; R_(z) is ahydrogen, amino, alkyl, aryl, arylalkyl, alkylamino, dialkylamino,alkylarylamino, hydrazine, or hydroxylalkylamino; and Q is oxygen,sulfur, methylene, imine, or alkylimine, provided that when R_(y) is2′-deoxy-D-ribose or D-ribose, R_(x) is not hydrogen and R_(z) is notamino.

The term “pharmaceutically-acceptable salts” embraces salts commonlyused to form alkali metal salts and to form addition salts of free acidsor free bases. The nature of the salt is not critical, provided that itis pharmaceutically-acceptable. Suitable pharmaceutically-acceptableacid addition salts of the compounds of the invention (preferably acompound of Formulas I-IV) may be prepared from an inorganic acid or anorganic acid. Examples of such inorganic acids are hydrochloric,hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic andsulfonic classes of organic acids, examples of which are formic, acetic,propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic),methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic,benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,cyclohexylaminosulfonic, stearic, algenic, .beta.-hydroxybutyric,malonic, galactic, and galacturonic acid. Suitablepharmaceutically-acceptable base addition salts of compounds of theinvention (preferably a compound of Formulas I-IV) include, but are notlimited to, metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine and procaine. All of these salts maybe prepared by conventional means from the corresponding compound of theinvention (preferably a compound of Formulas I-IV) by treating, forexample, the compound of the invention (preferably a compound ofFormulas I-IV) with the appropriate acid or base.

The invention also embraces isolated compounds. An isolated compoundrefers to a compound which represents at least 10%, preferably 20%, morepreferably 50% and most preferably 80% of the compound present in themixture, and exhibits a detectable (i.e. statistically significant)inhibitory activity of DNA methylation when tested in biological assayssuch as the combined bisulfite restriction analysis or COBRA (Xiong, Z.;Laird, P. W. Nucleic Acids Res. 1997, 25, 2532-2534) and radiolabeledmethyl incorporation assay (Francis, K. T.; Thompson, R. W.; Krumdieck,C. L. Am. J. Clin. Nutr. 1977, 30, 2028-2032)

3. Pharmaceutical Formulations of the Present Invention

According to the present invention, the cytosine analogs and derivativescan be formulated into pharmaceutically acceptable compositions fortreating various diseases and conditions.

The pharmaceutically-acceptable compositions of the present inventioncomprise one or more compounds of the invention (preferably compounds ofFormula I-IV) in association with one or more nontoxic,pharmaceutically-acceptable carriers and/or diluents and/or adjuvantsand/or excipients, collectively referred to herein as “carrier”materials, and if desired other active ingredients.

The compounds of the present invention (preferably compounds of FormulaI-IV) are administered by any route, preferably in the form of apharmaceutical composition adapted to such a route, as illustrated belowand are dependent on the condition being treated. The compounds andcompositions can be, for example, administered orally, parenterally,intraperitoneally, intravenously, intraarterially, transdernally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery (for example by a catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally.

The pharmaceutical formulation may optionally further include anexcipient added in an amount sufficient to enhance the stability of thecomposition, maintain the product in solution, or prevent side effects(e.g., potential ulceration, vascular irritation or extravasation)associated with the administration of the inventive formulation.Examples of excipients include, but are not limited to, mannitol,sorbitol, lactose, dextrox, cyclodextrin such as, α-, β-, andγ-cyclodextrin, and modified, amorphous cyclodextrin such ashydroxypropyl-, hydroxyethyl-, glucosyl-, maltosyl-, maltotriosyl-,carboxyamidomethyl-, carboxymethyl-, sulfobutylether-, anddiethylamino-substituted α-, β-, and γ-cyclodextrin. Cyclodextrins suchas Encapsin® from Janssen Pharmaceuticals or equivalent may be used forthis purpose.

For oral administration, the pharmaceutical compositions can be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica, or talc;disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs may contain conventional additives such as suspending agents,emulsifying agents, non-aqueous agents, preservatives, coloring agentsand flavoring agents. Examples of additives for liquid preparationsinclude acacia, almond oil, ethyl alcohol, fractionated coconut oil,gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin,methyl cellulose, methyl or propyl para-hydroxybenzoate, propyleneglycol, sorbitol, or sorbic acid.

For topical use the compounds of the present invention can also beprepared in suitable forms to be applied to the skin, or mucus membranesof the nose and throat, and can take the form of creams, ointments,liquid sprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compounds of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery.

The pharmaceutical compositions can be administered via injection.Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

In a particular embodiment, the compound of the present invention can beformulated into a pharmaceutically acceptable composition comprising thecompound solvated in non-aqueous solvent that includes glycerin,propylene glycol, polyethylene glycol, or combinations thereof. It isbelieved that the compound decitabine will be stable in suchpharmaceutical formulations so that the pharmaceutical formulations maybe stored for a prolonged period of time prior to use.

As discussed above, in current clinical treatment with decitabine, tominimize drug decomposition decitabine is supplied as lyophilized powderand reconstituted in a cold aqueous solution containing water in atleast 40% vol of the solvent, such as WFI, and diluted in cold infusionfluids prior to administration. Such a formulation and treatment regimensuffers from a few drawbacks. First, refrigeration of decitabine in coldsolution becomes essential, which is burdensome in handling andeconomically less desirable than a formulation that can sustain storageat higher temperatures. Second, due to rapid decomposition of decitabinein aqueous solution, the reconstituted decitabine solution may only beinfused to a patient for a maximum of 3 hr if the solution has beenstored in the refrigerator for less than 7 hr. In addition, infusion ofcold fluid can cause great discomfort and pain to the patient, whichinduces the patient's resistance to such a regimen.

By modifying the triazine ring and/or the ribose ring of decitabine andby formulating the compound with non-aqueous solvent, the pharmaceuticalformulations can circumvent the above-listed problems associated withthe current clinical treatment with decitabine. These formulations ofthe inventive compounds are believed to be more chemically stable thandecitabine formulated in aqueous solutions containing water in at least40% vol. of the solvent.

In a preferred embodiment, the inventive formulation contains less than40% water in the solvent, optionally less than 20% water in the solvent,optionally less than 10% water in the solvent, or optionally less than1% water in the solvent. In one variation, the pharmaceuticalformulation is stored in a substantially anhydrous form. Optionally, adrying agent may be added to the pharmaceutical formulation to absorbwater.

Owing to the enhanced stability, the inventive formulation may be storedand transported at ambient temperature, thereby significantly reducingthe cost of handling the drug. Further, the inventive formulation may beconveniently stored for a long time before being administered to thepatient. In addition, the inventive formulation may be diluted withregular infusion fluid (without chilling) and administered to a patientat room temperature, thereby avoiding causing patients' discomfortassociated with infusion of cold fluid.

In another embodiment, the inventive compound is dissolved in glycerinat different concentrations. For example, the formulation may optionallycomprise between 0.1 and 200; between 1 and 100; between 1 and 50;between 2 and 50; between 2 and 100; between 5 and 100; between 10 and100 or between 20 and 100 mg inventive compound per ml of glycerin.Specific examples of the inventive compound per glycerin concentrationsinclude but are not limited to 2, 5, 10, 20, 22, 25, 30, 40 and 50mg/ml.

Different grades of glycerin (synonyms: 1,2,3-propanetriol; glycerol;glycol alcohol; glycerol anhydrous) may be used to prepare theformulations. Preferably, glycerin with chemical purity higher than 90%is used to prepare the formulations.

In another embodiment, the inventive compound is dissolved in propyleneglycol at different concentrations. For example, the formulation mayoptionally comprise between 0.1 and 200; between 0.1 and 100; between0.1 and 50; between 2 and 50; between 2 and 100; between 5 and 100;between 10 and 100 or between 20 and 100 mg inventive compound per ml ofpropylene glycol. Specific examples of decitabine per propylene glycolconcentrations include but are not limited to 2, 5, 10, 20, 22, 25, 30,40 and 50 mg/ml.

In yet another embodiment, the inventive compound is dissolved in asolvent combining glycerin and propylene glycol at differentconcentrations. The concentration of propylene glycol in the solvent isbetween 0.1-99.9%, optionally between 1-90%, between 10-80%, or between50-70%.

In yet another embodiment, the inventive compound is dissolved atdifferent concentrations in a solvent combining glycerin andpolyethylene glycol (PEG) such as PEG300, PEG400 and PEG1000. Theconcentration of polyethylene glycol in the solvent is between0.1-99.9%, optionally between 1-90%, between 10-80%, or between 50-70%.

In yet another embodiment, the inventive compound is dissolved atdifferent concentrations in a solvent combining propylene glycol,polyethylene glycol and glycerin. The concentration of propylene glycolin the solvent is between 0.1-99.9%, optionally between 1-90%, between10-60%, or between 20-40%; and the concentration of polyethylene glycolin the solvent is between 0.1-99.9%, optionally between 1-90%, between10-80%, or between 50-70%.

It is believed and experimentally proven that addition of propyleneglycol can further improve chemical stability, reduce viscosity of theformulations and facilitate dissolution of the inventive compound in thesolvent.

The pharmaceutical formulation may further comprise an acidifying agentadded to the formulation in a proportion such that the formulation has aresulting pH between about 4 and 8. The acidifying agent may be anorganic acid. Examples of organic acid include, but are not limited to,ascorbic acid, citric acid, tartaric acid, lactic acid, oxalic acid,formic acid, benzene sulphonic acid, benzoic acid, maleic acid, glutamicacid, succinic acid, aspartic acid, diatrizoic acid, and acetic acid.The acidifying agent may also be an inorganic acid, such as hydrochloricacid, sulphuric acid, phosphoric acid, and nitric acid.

It is believed that adding an acidifying agent to the formulation tomaintain a relatively neutral pH (e.g., within pH 4-8) facilitates readydissolution of the inventive compound in the solvent and enhanceslong-term stability of the formulation. In alkaline solution, there is arapid reversible decomposition of decitabine toN-(formylamidino)-N′-β-D-2-deoxyribofuranosylurea, which decomposesirreversibly to form 1-β-D-2′-deoxyribofuranosyl-3-guanylurea. The firststage of the hydrolytic degradation involves the formation ofN-amidinium-N′-(2-deoxy-β-D-erythropentofuranosyl)urea formate (AUF).The second phase of the degradation at an elevated temperature involvesformation of guanidine. In acidic solution,N-(formylamidino)-N′-β-D-2-deoxyribofuranosylurea and some unidentifiedcompounds are formed. In strongly acidic solution (at pH<2.2)5-azacytosine is produced. Thus, maintaining a relative neutral pH maybe advantageous for the formulation comprising the analogs andderivatives of decitabine.

In a variation, the acidifying agent is ascorbic acid at a concentrationof 0.01-0.2 mg/ml of the solvent, optionally 0.04-0.1 mg/ml or 0.03-0.07mg/ml of the solvent.

The pH of the pharmaceutical formulation may be adjusted to be betweenpH 4 and pH 8, preferably between pH 5 and pH 7, and more preferablybetween pH 5.5 and pH 6.8.

The pharmaceutical formulation is preferably at least 80%, 90%, 95% ormore stable upon storage at 25° C. for 7, 14, 21, 28 or more days. Thepharmaceutical formulation is also preferably at least 80%, 90%, 95% ormore stable upon storage at 40° C. for 7, 14, 21, 28 or more days.

In one embodiment, the pharmaceutical formulation of the presentinvention is prepared by taking glycerin and dissolving the inventivecompound in the glycerin. This may be done, for example, by adding theinventive compound to the glycerin or by adding the glycerin todecitabine. By their admixture, the pharmaceutical formulation isformed.

Optionally, the method further comprises additional steps to increasethe rate at which the inventive compound is solvated by the glycerin.Examples of additional steps that may be performed include, but are norlimited to, agitation, heating, extension of solvation period, andapplication of micronized inventive compound and the combinationsthereof.

In one variation, agitation is applied. Examples of agitation includebut are nor limited to, mechanical agitation, sonication, conventionalmixing, conventional stirring and the combinations thereof. For example,mechanical agitation of the formulations may be performed according tomanufacturer's protocols by Silverson homogenizer manufactured bySilverson Machines Inc., (East Longmeadow, Mass.).

In another variation, heat may be applied. Optionally, the formulationsmay be heated in a water bath. Preferably, the temperature of the heatedformulations may be less than 70° C., more preferably, between 25° C.and 40° C. As an example, the formulation may be heated to 37° C.

In yet another variation, the inventive compound is solvated in glycerinover an extended period of time.

In yet another variation, a micronized form of the inventive compoundmay also be employed to enhance solvation kinetics. Optionally,micronization may be performed by a milling process. As an example,micronization may be performed by milling process performedMastersizerusing an Air Jet Mill, manufactured by IncFluid Energy AljetInc. (Boise, Id. Telford, Pa.).

Optionally, the method further comprises adjusting the pH of thepharmaceutical formulations by commonly used methods. In one variation,pH is adjusted by addition of acid, such as ascorbic acid, or base, suchas sodium hydroxide. In another variation, pH is adjusted and stabilizedby addition of buffered solutions, such as solution of(Ethylenedinitrilo) tetraacetic acid disodium salt (EDTA). As decitabineis known to be pH-sensitive, adjusting the pH of the pharmaceuticalformulations to approximately pH 7 may increase the stability oftherapeutic component.

Optionally, the method further comprises separation of non-dissolvedinventive compound from the pharmaceutical formulations. Separation maybe performed by any suitable technique. For example, a suitableseparation method may include one or more of filtration, sedimentation,and centrifugation of the pharmaceutical formulations. Clogging that maybe caused by non-dissolved particles of the inventive compound, maybecome an obstacle for administration of the pharmaceutical formulationsand a potential hazard for the patient. The separation of non-dissolvedinventive compound from the pharmaceutical formulations may facilitateadministration and enhance safety of the therapeutic product.

Optionally, the method further comprises sterilization of thepharmaceutical formulations. Sterilization may be performed by anysuitable technique. For example, a suitable sterilization method mayinclude one or more of sterile filtration, chemical, irradiation, heat,and addition of a chemical disinfectant to the pharmaceuticalformulation.

As noted, decitabine is unstable in water and hence it may be desirableto reduce the water content of the glycerin used for formulating theinventive compound. Accordingly, prior to the dissolution and/orsterilization step, the glycerin may be dried. Such drying of glycerinor the solution of the inventive compound in glycerin may be achieved bythe addition of a pharmaceutically acceptable drying agent to theglycerin. The glycerin or the inventive formulations may be dried, forexample by filtering it through a layer comprising a drying agent.

Optionally, the method may further comprise adding one or more membersof the group selected from drying agents, buffering agents,antioxidants, stabilizers, antimicrobials, and pharmaceutically inactiveagents. In one variation, antioxidants such as ascorbic acid, ascorbatesalts and mixtures thereof may be added. In another variationstabilizers like glycols may be added.

4. Vessels or Kits Containing Inventive Compounds or Formulations

The pharmaceutical formulations, described in this invention, may becontained in a sterilized vessel such as syringes, vials or ampoules ofvarious sizes and capacities. The sterilized vessel may optionallycontain between 1-50 ml, 1-25 ml or 1-20 ml or 1-10 ml of theformulations. Sterilized vessels maintain sterility of thepharmaceutical formulations, facilitate transportation and storage, andallow administration of the pharmaceutical formulations without priorsterilization step.

The present invention also provides a kit for administering theinventive compound to a host in need thereof. In one embodiment, the kitcomprises the inventive compound in a solid, preferably powder form, anda non-aqueous diluent that comprises glyercin, propylene glycol,polyethylene glycol, or combinations thereof. Mixing of the soliddecitabine and the diluent preferably results in the formation of apharmaceutical formulation according to the present invention. Forexample, the kit may comprise a first vessel comprising the inventivecompound in a solid form; and a vessel container comprising a diluentthat comprises glyercin; wherein adding the diluent to the solidinventive compound results in the formation of a pharmaceuticalformulation for administering the inventive compound. Mixing the solidthe inventive compound and diluent may optionally form a pharmaceuticalformulation that comprises between 0.1 and 200 mg of the inventivecompound per ml of the diluent, optionally between 0.1 and 100, between2 mg and 50 mg, 5 mg and 30 mg, between 10 mg and 25 mg per ml of thesolvent.

According to the embodiment, the diluent is a combination of propyleneglycol and glycerin, wherein the concentration of propylene glycol inthe solvent is between 0.1-99.9%, optionally between 1-90%, between10-60%, or between 20-40%.

Also according to the embodiment, the diluent is a combination ofpolyethylene glycol and glycerin, wherein the concentration ofpolyethylene glycol in the solvent is between 0.1-99.9%, optionallybetween 1-90%, between 10-60%, or between 20-40%.

Also according to the embodiment, the diluent is a combination ofpropylene glycol, polyethylene glycol and glycerin, wherein theconcentration of propylene glycol in the solvent is between 0.1-99.9%,optionally between 1-90%, between 10-60%, or between 20-40%; and theconcentration of polyethylene glycol in the solvent is between0.1-99.9%, optionally between 1-90%, between 10-60%, or between 20-40%.

The diluent also optionally comprises 40%, 20%, 10%, 5%, 2% or lesswater. In one variation, the diluent is anhydrous and may optionallyfurther comprise a drying agent. The diluent may also optionallycomprise one or more drying agents, glycols, antioxidants and/orantimicrobials.

The kit may optionally further include instructions. The instructionsmay describe how the solid the inventive compound and the diluent shouldbe mixed to form a pharmaceutical formulation. The instructions may alsodescribe how to administer the resulting pharmaceutical formulation to apatient. It is noted that the instructions may optionally describe theadministration methods according to the present invention.

The diluent and the inventive compound may be contained in separatevessels. The vessels may come in different sizes. For example, thevessel may comprise between 1 and 50, 1 and 25, 1 and 20, or 1 and 10 mlof the diluent.

The pharmaceutical formulations provided in vessels or kits may be in aform that is suitable for direct administration or may be in aconcentrated form that requires dilution relative to what isadministered to the patient. For example, pharmaceutical formulations,described in this invention, may be in a form that is suitable fordirect administration via infusion.

The methods and kits described herein provide flexibility whereinstability and therapeutic effect of the pharmaceutical formulationscomprising the inventive compound may be further enhanced orcomplemented.

5. Methods for Administrating Inventive Compounds/Compositions

The compounds/formulations of the present invention can be administeredby any route, preferably in the form of a pharmaceutical compositionadapted to such a route, as illustrated below and are dependent on thecondition being treated. The compounds or formulations can be, forexample, administered orally, parenterally, topically,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery (for example by catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally. The compoundsand/or compositions according to the invention may also be administeredor co-administered in slow release dosage forms.

The compounds and/or compositions of this invention may be administeredor co-administered in any conventional dosage form. Co-administration inthe context of this invention is defined to mean the administration ofmore than one therapeutic agent in the course of a coordinated treatmentto achieve an improved clinical outcome. Such co-administration may alsobe coextensive, that is, occurring during overlapping periods of time.

The inventive compound or the composition containing the inventivecompound may be administered into a host such as a patient at a dose of0.1-1000 mg/m², optionally 1-200 mg/m², optionally 1-50 mg/m²,optionally 1-40 mg/m², optionally 1-30 mg/m², optionally 1-20 mg/m², oroptionally 5-30 mg/m².

For example, the compound/composition of the present invention may besupplied as sterile powder for injection, together with buffering saltsuch as potassium dihydrogen and pH modifier such as sodium hydroxide.This formulation is preferably stored at 2-8° C., which should keep thedrug stable for at least 2 years. This powder formulation may bereconstituted with 10 ml of sterile water for injection. This solutionmay be further diluted with infusion fluid known in the art, such as0.9% sodium chloride injection, 5% dextrose injection and lactatedringer's injection. It is preferred that the reconstituted and dilutedsolutions be used within 4-6 hours for delivery of maximum potency.

In a preferred embodiment, the inventive compound/composition isadministered to a patient by injection, such as subcutaneous injection,bolus i.v. injection, continuous i.v. infusion and i.v. infusion over 1hour. Optionally the inventive compound/composition is administered to apatient via an 1-24 hour i.v. infusion per day for 3-5 days pertreatment cycle at a dose of 0. 1-1000 mg/m² per day, optionally at adose of 1-100 mg/m² per day, optionally at a dose of 2-50 mg/m² per day,optionally at a dose of 10-30 mg/m² per day, or optionally at a dose of5-20 mg/m² per day,

For decitabine or azacitidine, the dosage below 50 mg/m² is consideredto be much lower than that used in conventional chemotherapy for cancer.By using such a low dose of the analog/derivative of decitabine orazacitidine, transcriptional activity of genes silenced in the cancercells by aberrant methylation can be activated to trigger downstreamsignal transduction, leading to cell growth arrest, differentiation andapoptosis, which eventually results in death of these cancer cells. Thislow dosage, however, should have less systemic cytotoxic effect onnormal cells, and thus have fewer side effects on the patient beingtreated.

The pharmaceutical formulations may be co-administered in anyconventional form with one or more member selected from the groupcomprising infusion fluids, therapeutic compounds, nutritious fluids,anti-microbial fluids, buffering and stabilizing agents.

As described above, the inventive compounds can be formulated in aliquid form by solvating the inventive compound in a non-aqueous solventsuch as glycerin. The pharmaceutical liquid formulations provide thefurther advantage of being directly administrable, (e.g., withoutfurther dilution) and thus can be stored in a stable form untiladministration. Further, because glycerin can be readily mixed withwater, the formulations can be easily and readily further diluted justprior to administration. For example, the pharmaceutical formulationscan be diluted with water 180, 60, 40, 30, 20, 10, 5, 2, 1 minute orless before administration to a patient.

Patients may receive the pharmaceutical formulations intravenously. Thepreferred route of administration is by intravenous infusion.Optionally, the pharmaceutical formulations of the current invention maybe infused directly, without prior reconstitution.

In one embodiment, the pharmaceutical formulation is infused through aconnector, such as a Y site connector, that has three arms, eachconnected to a tube. As an example, Baxter® Y-connectors of varioussizes can be used. A vessel containing the pharmaceutical formulation isattached to a tube further attached to one arm of the connector.Infusion fluids, such as 0.9% sodium chloride, or 5% dextrose, or 5%glucose, or Lactated Ringer's, are infused through a tube attached tothe other arm of the Y-site connector. The infusion fluids and thepharmaceutical formulations are mixed inside the Y site connector. Theresulting mixture is infused into the patient through a tube connectedto the third arm of the Y site connector. The advantage of thisadministration approach over the prior art is that the inventivecompound is mixed with infusion fluids before it enters the patient'sbody, thus reducing the time when decomposition of the inventivecompound may occur due to contact with water. For example, the inventivecompound is mixed less than 10, 5, 2 or 1 minutes before entering thepatient's body.

Patients may be infused with the pharmaceutical formulations for 1, 2,3, 4, 5 or more hours, as a result of the enhanced stability of theformulations. Prolonged periods of infusion enable flexible schedules ofadministration of therapeutic formulations.

Alternatively or in addition, speed and volume of the infusion can beregulated according to the patient's needs. The regulation of theinfusion of the pharmaceutical formulations can be performed accordingto existing protocols.

The pharmaceutical formulations may be co-infused in any conventionalform with one or more member selected from the group comprising infusionfluids, therapeutic compounds, nutritious fluids, anti-microbial fluids,buffering and stabilizing agents. Optionally, therapeutic componentsincluding, but are not limited to, anti-neoplastic agents, alkylatingagents, agents that are members of the retinoids superfamily, antibioticagents, hormonal agents, plant-derived agents, biologic agents,interleukins, interferons, cytokines, immuno-modulating agents, andmonoclonal antibodies, may be co-infused with the inventiveformulations.

Co-infusion in the context of this invention is defined to mean theinfusion of more than one therapeutic agents in a course of coordinatedtreatment to achieve an improved clinical outcome. Such co-infusion maybe simultaneous, overlapping, or sequential. In one particular example,co-infusion of the pharmaceutical formulations and infusion fluids maybe performed through Y-type connector.

The pharmacokinetics and metabolism of intravenously administered thepharmaceutical formulations resemble the pharmacokinetics and metabolismof intravenously administered the inventive compound.

In humans, decitabine displayed a distribution phase with a half-life of7 minutes and a terminal half-life on the order of 10-35 minutes asmeasured by bioassay. The volume of distribution is about 4.6 L/kg. Theshort plasma half-life is due to rapid inactivation of decitabine bydeamination by liver cytidine deaminase. Clearance in humans is high, onthe order of 126 mL/min/kg. The mean area under the plasma curve in atotal of 5 patients was 408 μg/h/L with a peak plasma concentration of2.01 μM. In patients decitabine concentrations were about 0.4 μg/ml (2μM) when administered at 100 mg/m² as a 3-hour infusion. During a longerinfusion time (up to 40 hours) plasma concentration was about 0.1 to 0.4μg/mL. With infusion times of 40-60 hours, at an infusion rate of 1mg/kg/h, plasma concentrations of 0.43-0.76 μg/mL were achieved. Thesteady-state plasma concentration at an infusion rate of 1 mg/kg/h isestimated to be 0.2-0.5 μg/mL. The half-life after discontinuing theinfusion is 12-20 min. The steady-state plasma concentration ofdecitabine was estimated to be 0.31-0.39 μg/mL during a 6-hour infusionof 100 mg/m². The range of concentrations during a 600-mg/m² infusionwas 0.41-16 μg/mL. Penetration of decitabine into the cerebrospinalfluid in man reaches 14-21% of the plasma concentration at the end of a36-hour intravenous infusion. Urinary excretion of unchanged decitabineis low, ranging from less than 0.01% to 0.9% of the total dose, andthere is no relationship between excretion and dose or plasma druglevels. High clearance values and a total urinary excretion of less than1% of the administered dose suggest that decitabine is eliminatedrapidly and largely by metabolic processes.

Owing to their enhanced stability in comparison with decitabine, theinventive compounds/compositions can enjoy longer shelf life when storedand circumvent problems associated with clinical use of decitabine. Forexample, the inventive compounds may be supplied as lyophilized powder,optionally with an excipient (e.g., cyclodextrin), acid (e.g., ascorbicacid), alkaline (sodium hydroxide), or buffer salt (monobasic potassiumdihydrogen phosphate). The lyophilized powder can be reconstituted withsterile water for injection, e.g., i.v., i.p., i.m., or subcutaneously.Optionally, the powder can be reconstituted with aqueous or non-aqueoussolvent comprising a water miscible solvent such as glycerin, propyleneglycol, ethanol and PEG. The resulting solution may be administereddirectly to the patient, or diluted further with infusion fluid, such as0.9% Sodium Chloride; 5% Dextrose; 5% Glucose; and Lactated Ringer'sinfusion fluid.

The inventive compounds/compositions may be stored under ambientconditions or in a controlled environment, such as under refrigeration(2-8° C.; 36-46° F.). Due to their superior stability in comparison withdecitabine, the inventive compounds/compositions can be stored at roomtemperature, reconstituted with injection fluid, and administered to thepatient without prior cooling of the drug solution.

In addition, due to their enhanced chemical stability, the inventivecompound/composition should have a longer plasma half-life compared tothat of decitabine. Thus, the inventive compound/composition may beadministered to the patient at a lower dose and/or less frequently thanthat for decitabine.

6. Combination Therapy with Inventive Pharmaceutical Compositions

The pharmaceutical formulations of the present invention may be used inconjunction with therapeutic components including but not limiting toanti-neoplastic agents, alkylating agents, agents that are members ofthe retinoids superfamily, antibiotic agents, hormonal agents,plant-derived agents, biologic agents, interleukins, interferons,cytokines, immuno-modulating agents, and monoclonal antibodies.

In one embodiment, an alkylating agent is used in combination withand/or added to the inventive compound/formulation. Examples ofalkylating agents include, but are not limited to bischloroethylamines(nitrogen mustards, e.g. chlorambucil, cyclophosphamide, ifosfamide,mechlorethamine, melphalan, uracil mustard), aziridines (e.g. thiotepa),alkyl alkone sulfonates (e.g. busulfan), nitrosoureas (e.g. carmustine,lomustine, streptozocin), nonclassic alkylating agents (altretamine,dacarbazine, and procarbazine), platinum compounds (carboplastin andcisplatin).

In another embodiment, cisplatin, carboplatin or cyclophosphamide isused in combination with and/or added to the inventivecompound/formulation.

In another embodiment, a member of the retinoids superfamily is used incombination with and/or added to the inventive compound/formulation.Retinoids are a family of structurally and functionally relatedmolecules that are derived or related to vitamin A (all-trans-retinol).Examples of retinoid include, but are not limited to, all-trans-retinol,all-trans-retinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin)and 9-cis-retinoic acid.

In yet another embodiment, a hormonal agent is used in combination withand/or added to the inventive compound/formulation. Examples of such ahormonal agent are synthetic estrogens (e.g. diethylstibestrol),antiestrogens (e.g. tamoxifen, toremifene, fluoxymesterol andraloxifene), antiandrogens (bicalutamide, nilutamide, flutamide),aromatase inhibitors (e.g., aminoglutethimide, anastrozole andtetrazole), ketoconazole, goserelin acetate, leuprolide, megestrolacetate and mifepristone.

In yet another embodiment, a plant-derived agent is used in combinationwith and/or added to the inventive compound/formulation. Examples ofplant-derived agents include, but are not limited to, vinca alkaloids(e.g., vincristine, vinblastine, vindesine, vinzolidine andvinorelbine), camptothecin (20(S)-camptothecin,9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin),podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), andtaxanes (e.g., paclitaxel and docetaxel).

In yet another embodiment, a biologic agent is used in combination withand/or added to the inventive compound/formulation, such asimmuno-modulating proteins such as cytokines, monoclonal antibodiesagainst tumor antigens, tumor suppressor genes, and cancer vaccines.

Examples of interleukins that may be used in combination with and/oradded to the inventive compound/formulation include, but are not limitedto, interleukin 2 (IL-2), and interleukin 4 (IL-4), interleukin 12(IL-12). Examples of interferons that may be used in conjunction withdecitabine-glycerin formulations include, but are not limited to,interferon α, interferon β (fibroblast interferon) and interferon γ(fibroblast interferon). Examples of such cytokines include, but are notlimited to erythropoietin (epoietin), granulocyte-CSF (filgrastim), andgranulocyte, macrophage-CSF (sargramostim). Immuno-modulating agentsother than cytokines include, but are not limited to bacillusCalmette-Guerin, levamisole, and octreotide.

Example of monoclonal antibodies against tumor antigens that can be usedin conjunction with the inventive formulations include, but are notlimited to, HERCEPTIN® (Trastruzumab), RITUXAN® (Rituximab), MYLOTARG®(anti-CD33), and CAMPATH® (anti-CD52).

7. Indications for Compounds or Pharmaceutical Compositions of thePresent Invention

The pharmaceutical formulations according to the present invention maybe used to treat a wide variety of diseases associated with aberrant DNAmethylation, or those sensitive to the treatment with decitabine.

Many diseases are associated with aberrant DNA methylation, especiallytumors, cancers and hematological disorders. See Esteller, M., A Gene“Hypermethylation Profile of Human Cancer,” Cancer Research, (2001)61:3225, 3229 (associating colon cancer, stomach cancer, pancreaticcancer, liver cancer, kidney cancer, lung cancer, head and neck cancer,breast cancer, ovarian cancer, endometrium cancer, bladder cancer, braincancer, leukemia, and lymphomas with DNA hypermethylation); Santini, V.,et al., “Changes in DNA Methylation in Neoplasia: Pathophysiology andTherapeutic Implications,” Annals of Internal Medicine, (2001)134:573-586 (associating colon cancer, breast cancer, gastric cancer,endometrial cancer, retinoblastoma, renal-cell cancer, ovarian cancer,lung cancer, melonoma, mesothelioma, acute myelogenous leukemia,myelodysplastic syndromes, chronic meylogenous leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma,lymphoma, prostate cancer, chronic meylogenous leukemia, prostatecancer, esophageal cancer, acute leukemia, malignant hematologicdisease, glioblastoma multiforme with DNA hypermethylation); Baylin, S.B., et al., “Alterations in DNA Methylation: A fundamental Aspect ofNeoplasia,” Cancer Res., (1998) 72: 141-196 (associating retinoblastoma,renal carcinoma, solid tumors, lymphomas, primary acute leukemias,Burkitt lymphoma, bladder cancer, breast cancer, colon cancer, livercancer, lung cancer, leukemia, brain cancer, renal cancer, prostatecancer, and carcinomas with DNA hypermethylation); Wajed, S. A., “DNAMethylation: An Alternative Pathway to Cancer,” Annals of Surgery,(2001) Vol. 234, No. 1, 10-20 (associating esophagus, gastric,colorectal, pancreas, lung, bladder, ovary, breast melanoma, leukemia,gastric, endometrium, ovary, thyroid, kidney, brain, colon, prostatewith DNA hypermethylation); Esteller, M., “Epigenetic Lesions CausingGenetic Lesions in human Cancer: Promoter Hypermethylation of DNA RepairGenes,” European Journal of Cancer, (2000) 3:2294-2300 associating braincancer, head and neck cancer, lymphomas, lung cancer, breast cancer,kidney cancer, stomach cancer, colon cancer, liver cancer, cancer of theuterus, and prostate cancer to DNA hypermethylation); and Esteller, M.,CpG Island “Hypermethylation And Tumor Suppressor Genes: A BoomingPresent, A Brighter Future,” Oncogene, (2002) 21:5427-5440 (associatinghemangioblastoma, retinoblastoma, glioma, stomach cancer, leukemia,lymphoma, etc. with DNA hypermethylation).

Preferable indications that may be treated using the pharmaceuticalformulations of the present invention include those involvingundesirable or uncontrolled cell proliferation. Such indications includebenign tumors, various types of cancers such as primary tumors and tumormetastasis, restenosis (e.g. coronary, carotid, and cerebral lesions),hematological disorders, abnormal stimulation of endothelial cells(atherosclerosis), insults to body tissue due to surgery, abnormal woundhealing, abnormal angiogenesis, diseases that produce fibrosis oftissue, repetitive motion disorders, disorders of tissues that are nothighly vascularized, and proliferative responses associated with organtransplants.

Generally, cells in a benign tumor retain their differentiated featuresand do not divide in a completely uncontrolled manner. A benign tumor isusually localized and nonmetastatic. Specific types benign tumors thatcan be treated using the present invention include hemangiomas,hepatocellular adenoma, cavernous haemangioma, focal nodularhyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bileduct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas and pyogenicgranulomas.

In a malignant tumor cells become undifferentiated, do not respond tothe body's growth control signals, and multiply in an uncontrolledmanner. The malignant tumor is invasive and capable of spreading todistant sites (metastasizing). Malignant tumors are generally dividedinto two categories: primary and secondary. Primary tumors arisedirectly from the tissue in which they are found. A secondary tumor, ormetastasis, is a tumor which is originated elsewhere in the body but hasnow spread to a distant organ. The common routes for metastasis aredirect growth into adjacent structures, spread through the vascular orlymphatic systems, and tracking along tissue planes and body spaces(peritoneal fluid, cerebrospinal fluid, etc.)

Specific types of cancers or malignant tumors, either primary orsecondary, that can be treated using this invention include breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gall bladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet celltumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullarycarcinoma, pheochromocytoma, mucosal neuronms, intestinalganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

Hematologic disorders include abnormal growth of blood cells which canlead to dysplastic changes in blood cells and hematologic malignanciessuch as various leukemias. Examples of hematologic disorders include butare not limited to acute myeloid leukemia, acute promyelocytic leukemia,acute lymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes, and sickle cell anemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchromosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subgroup ofAML. This subtype is characterized by promyelocytic blasts containingthe 15;17 chromosomal translocation. This translocation leads to thegeneration of the fusion transcript comprised of the retinoic acidreceptor and a sequence PML.

Acute lymphoblastic leukemia (ALL) is a heterogenerous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality is the 9;22 translocation. The resultantPhiladelphia chromosome represents poor prognosis of the patient.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferativedisorder of a pluripotent stem cell. CML is characterized by a specificchromosomal abnormality involving the translocation of chromosomes 9 and22, creating the Philadelphia chromosome. Ionizing radiation isassociated with the development of CML.

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia.

Treatment of abnormal cell proliferation due to insults to body tissueduring surgery may be possible for a variety of surgical procedures,including joint surgery, bowel surgery, and cheloid scarring. Diseasesthat produce fibrotic tissue include emphysema. Repetitive motiondisorders that may be treated using the present invention include carpaltunnel syndrome. An example of cell proliferative disorders that may betreated using the invention is a bone tumor.

The proliferative responses associated with organ transplantation thatmay be treated using this invention include those proliferativeresponses contributing to potential organ rejections or associatedcomplications. Specifically, these proliferative responses may occurduring transplantation of the heart, lung, liver, kidney, and other bodyorgans or organ systems.

Abnormal angiogenesis that may be may be treated using this inventioninclude those abnormal angiogenesis accompanying rheumatoid arthritis,ischemic-reperfusion related brain edema and injury, cortical ischemia,ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome),endometriosis, psoriasis, diabetic retinopaphy, and other ocularangiogenic diseases such as retinopathy of prematurity (retrolentalfibroplastic), muscular degeneration, corneal graft rejection,neuroscular glaucoma and Oster Webber syndrome.

Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanism ofvarious angiogenic factors, including elements of the inflammatoryresponse, such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

In another embodiment, the pharmaceutical formulations of the presentinvention may be used for treating diseases associated with undesired orabnormal angiogenesis. The method comprises administering to a patientsuffering from undesired or abnormal angiogenesis the pharmaceuticalformulations of the present invention alone, or in combination withanti-neoplastic agent whose activity as an anti-neoplastic agent in vivois adversely affected by high levels of DNA methylation. The particulardosage of these agents required to inhibit angiogenesis and/orangiogenic diseases may depend on the severity of the condition, theroute of administration, and related factors that can be decided by theattending physician. Generally, accepted and effective daily doses arethe amount sufficient to effectively inhibit angiogenesis and/orangiogenic diseases.

According to this embodiment, the pharmaceutical formulations of thepresent invention may be used to treat a variety of diseases associatedwith undesirable angiogenesis such as retinal/choroidalneuvascularization and corneal neovascularization. Examples ofretinal/choroidal neuvascularization include, but are not limited to,Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease,vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis,pseudoxanthoma elasticum carotid abostructive diseases, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, diabeticretinopathy, macular degeneration, Bechets diseases, infections causinga retinitis or chroiditis, presumed ocular histoplasmosis, parsplanitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications, diseases associatedwith rubesis (neovascularization of the angle) and diseases caused bythe abnormal proliferation of fibrovascular or fibrous tissue includingall forms of proliferative vitreoretinopathy. Examples of cornealneuvascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, polyarteritis,Wegener sarcoidosis, Scleritis, periphigoid radial keratotomy,neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections and Kaposi sarcoma.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used for treating chronic inflammatory diseasesassociated with abnormal angiogenesis. The method comprisesadministering to a patient suffering from a chronic inflammatory diseaseassociated with abnormal angiogenesis the pharmaceutical formulations ofthe present invention alone, or in combination with an anti-neoplasticagent whose activity as an anti-neoplastic agent in vivo is adverselyaffected by high levels of DNA methylation. The chronic inflammationdepends on continuous formation of capillary sprouts to maintain aninflux of inflammatory cells. The influx and presence of theinflammatory cells produce granulomas and thus, maintains the chronicinflammatory state. Inhibition of angiogenesis using the pharmaceuticalformulations of the present invention may prevent the formation of thegranulosmas, thereby alleviating the disease. Examples of chronicinflammatory disease include, but are not limited to, inflammatory boweldiseases such as Crohn's disease and ulcerative colitis, psoriasis,sarcoidois, and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis are characterized by chronic inflammation and angiogenesis atvarious sites in the gastrointestinal tract. For example, Crohn'sdisease occurs as a chronic transmural inflammatory disease that mostcommonly affects the distal ileum and colon but may also occur in anypart of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea. Theseinflammatory bowel diseases are generally caused by chronicgranulomatous inflammation throughout the gastrointestinal tract,involving new capillary sprouts surrounded by a cylinder of inflammatorycells. Inhibition of angiogenesis by the pharmaceutical formulations ofthe present invention should inhibit the formation of the sprouts andprevent the formation of granulomas. The inflammatory bowel diseasesalso exhibit extra intestinal manifectations, such as skin lesions. Suchlesions are characterized by inflammation and angiogenesis and can occurat many sites other the gastrointestinal tract. Inhibition ofangiogenesis by the pharmaceutical formulations of the present inventionshould reduce the influx of inflammatory cells and prevent the lesionformation.

Sarcoidois, another chronic inflammatory disease, is characterized as amulti-system granulomatous disorder. The granulomas of this disease canform anywhere in the body and, thus, the symptoms depend on the site ofthe granulomas and whether the disease is active. The granulomas arecreated by the angiogenic capillary sprouts providing a constant supplyof inflammatory cells. By using the pharmaceutical formulations of thepresent invention to inhibit angionesis, such granulomas formation canbe inhibited. Psoriasis, also a chronic and recurrent inflammatorydisease, is characterized by papules and plaques of various sizes.Treatment using the pharmaceutical formulations of the present inventionshould prevent the formation of new blood vessels necessary to maintainthe characteristic lesions and provide the patient relief from thesymptoms.

Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Treatment using thepharmaceutical formulations of the present invention alone or inconjunction with other anti-RA agents may prevent the formation of newblood vessels necessary to maintain the chronic inflammation and providethe RA patient relief from the symptoms.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used for treating diseases associated withabnormal hemoglobin synthesis. The method comprises administering thepharmaceutical formulations of the present invention to a patientsuffering from disease associated with abnormal hemoglobin synthesis.Decitabine containing formulations stimulate fetal hemoglobin synthesisbecause the mechanism of incorporation into DNA is associated with DNAhypomethylation. Examples of diseases associated with abnormalhemoglobin synthesis include, but are not limited to, sickle cell anemiaand β-thalassemia.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used to control intracellular gene expression.The method comprises administering the pharmaceutical formulations ofthe present invention to a patient suffering from disease associatedwith abnormal levels of gene expression. DNA methylation is associatedwith the control of gene expression. Specifically, methylation in ornear promoters inhibit transcription while demethylation restoresexpression. Examples of the possible applications of the describedmechanisms include, but are not limited to, therapeutically modulatedgrowth inhibition, induction of apoptosis, and cell differentiation.

Gene activation facilitated by the pharmaceutical formulations of thepresent invention may induce differentiation of cells for therapeuticpurposes. Cellular differentiation is induced through the mechanism ofhypomethylation. Examples of morphological and functionaldifferentiation include, but are not limited to differentiation towardsformation of muscle cells, myotubes, cells of erythroid and lymphoidlineages.

Although exemplary embodiments of the present invention have beendescribed and depicted, it will be apparent to the artisan of ordinaryskill that a number of changes, modifications, or alterations to theinvention as described herein may be made, none of which depart from thespirit of the present invention. All such changes, modifications, andalterations should therefore be seen as within the scope of the presentinvention.

EXAMPLE

The following are examples of the compounds and methods of synthesisthereof according to the present invention.

1. Derivatives of Decitabine or Azacitidine with 4- and 6-PositionSubstitution

In this example, the analogs of decitabine or azacitidine modified atthe 4- and 6-position are described. As discussed in detail above, basedon their resonance analysis of decitabine and azacitidine the inventorsbelieve that introduction of a chemical group into certain position ofdecitabine is expected to increase aqueous stability of decitabine.

For example, as shown in FIG. 4A, electron-donating groups (+I) candecrease electrophilicity of the 6-position. Replacement of the6-position hydrogen (R_(x)) of 5-azacytidine with a field-effectelectron-donating group (+I) lowers the surrounding electrophilicity andprevents hydrolytic cleavage at the 6-position of the triazine ring.Similarly, substitution of the 4-position (R_(z)) with varyingelectron-donating groups (NH₂, NR₂>OH, OR>halogen>alkyl>H) reduceselectrophilicity of the 6-position, while electron-withdrawing groups(NO₂, NR₃ ⁺, CF₃, Cl₃>HC═O, R—C═O, CO₂H, CO₂R, SO₃H, CN>H) increases theelectrophilicity of the 6-position.

While the 6-position can be modified by any electron-donating alkylgroup, it is desirable to stabilize the 6-position against hydrolysiswithout interfering with the biological activity of decitabine. As thelength and size of the alkyl increase (FIG. 4B), it is possible that thedecitabine analogs may exist in conformations that are not favorable forbinding with DNA methyltransferase. Due to steric crowding, the alkylgroup may limit the analogs to certain conformations, and those may notnecessarily be favorable for binding with DNA methyltransferase.Therefore, it is preferred that a compromise is made between stabilityagainst hydrolysis and binding affinity toward DNA methyltransferase.FIG. 4B and FIG. 4C list the preferred alkyl derivatives of decitabineand azacitidine, respectively, that are made by substituting thehydrogen at the 6-position with a CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃,and phenyl group.

Synthesis of these alkyl-derivatives of decitabine can be achieved byusing a modified procedure based on Pliml, J. et al. Pliml, J.; Sorm, F.Collect. Czech. Chem. Commun. 1964, 29, 2576-2577. Specifically thePliml synthesis procedure can be modified at the cyclization of1-(2-deoxy-3,5-di-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiuret(1i, 4E) to yield the alkyl derivatives of decitabine. FIG. 4Dillustrates this modification, which gives decitabine derivatives 2, 3,4, 5 and 6 after deprotection.

In one embodiment, for example, azacytidine derivatives 2, 3, 4, 5 or 6was prepared by stirring and refluxing (˜90° C.) a mixture of1′-(2′-Deoxy-3′5′-di-O-p-chlorobenzoyl-D-ribofuranosyl)-4—O-methylisobiruet(1i) in formic acid (1.5 eq.) and 50 equivalents of trimethylorthoacetate, triethyl orthopropionate, trimethyl orthobutyrate,trimethyl orthovalerate, or trimethyl orthobenzoate, respectively, untilcomplete reaction (<5% 1i remaining) as determined by HPLC. The reactionwas cooled to 22±5° C. before charging slowly (in a period of not lessthan 2 hours) with water (not less than 3 kg of water per mole of 1iused) to precipitate the respective fully protected intermediate 2i, 3i,4i, 5 or 6i (FIGS. 4B and 4C). The mixture was stirred for an additionalone hour before it was cooled to 5±5° C. and stirred for two hours. Theslurry was filtered and the solid cake was dried in vacuo at 50±5° C.until loss on drying (LOD) was less than 0.1%. The dried intermediatewas suspended in a mixture of anhydrous methanol (˜21 liters per mole ofintermediate used) and anhydrous ammonia gas, dimethylamine, hydrazine,methylamine, ethylamine, propylamine or benzylamine (˜22 eq.), and themixture was stirred for not less than 48 hours and until completeconsumption of intermediate product. After reaction completion excessvolatile reagent such as ammonia and dimethylamine was removed byapplication of a slight vacuum for one hour before the mixture wasconcentrated in vacuo at 50±5° C. to a minimal volume (˜6 liters permole of intermediate used) and cool to 5±5° C. initiate crystallizationof the crude 2b-h, 3b-h, 4b-h, 52b-h or 6b-h. The crude product wasfiltered after 1 hour, washed with cold (5° C.) methanol (0.5 liter permole of intermediate used), re-dissolved in hot (42° C.) anhydrousmethanol (33 liters per mole of intermediate used), decolorized withactivated carbon (equivalent to 24 g per mole of intermediate used),filtered to obtain a clear solution of product, concentrated in vacuo atnot more than 50° C. to a minimal volume (˜2 liters mole per ofintermediate used), and allowed to crystallize at 22±5° C. for ˜12hours. The slurry was filtered, the crystalline product washed withmethanol (0.2 liter per mole of intermediate used), and the pure product2b-h, 3b-h, 4b-h, 5b-h or 6b-h dried in vacuo at not more than 40° C.for ˜8 hours.

Azacitidine derivatives 7b-h, 8b-h, 9b-h, 10b-h and 11b-h can likewisebe synthesized starting from a similarly protected ribose derivativesuch as1′-(2′,3′,5′-tri-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiruetvia intermediates 7i, 8i, 9i, 10i and 11i prior to deprotection (FIG.4C).

Decitabine derivatives 1′b-g and azacitidine derivatives 1″b-g canlikewise be synthesized starting from a similarly protected ribosederivative such as1′-(2′,3′,5′-tri-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiruetvia intermediates 1′i and 1″i (FIG. 4D), respectively.

Decitabine derivatives 1′a, 1″a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a and11a can be synthesized from the respective 1′i, 1″i, 2i, 3i, 4i, 5i, 6i,7i, 8i, 9i, 10i and 11i by treating with sodium methoxide or tertiaryamine (which includes but is not limited to triethylamine, ethyldiisopropylamine and DBU) to remove the 3′,5′-di-O-p-chlorobenzoylprotection without affecting the 4-OMe group.

2. Keto-Enol Derivatives of Decitabine or Azacitidine at the 6-Position

In this example, the N═CH functional group of the triazine ring ofdecitabine or azacitidine is replaced with a more stable NH—C═O⇄N═C—OHmoiety, producing keto-enol derivatives 12 and 13 are obtained (FIG.5A). This moiety is resistant to hydrolytic cleavage in aqueousconditions. In a basic medium where decitabine is easily hydrolyzed, theOH group of the keto-enol analog becomes the strong electron-donating(+I) group O⁻, which will prevent hydrolytic cleavage of the triazinering.

The keto-enol derivative of decitabine or azacytiding can be synthesizedby cyclizing1-(2-deoxy-3,5-di-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiuret(1i) with one-third equivalent of triphosgene (or one-half equivalent ofoxalyl chloride) to give the keto-enol 12 after deprotection (FIG. 5B).The azacytidine derivative 13 can likewise be synthesized.

In one embodiment, for example,1′-(2′-deoxy-3′,5′-di-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiuret(1i) or1′-(2′,3′,5′-tri-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiruet(1j) was dissolved (0.001 to 10 M concentration) in an anhydrous aproticorganic solvent effective in solubilizing it. These solvents include,but are not limited to: acetonitrile; chlorobenzene; dichloromethane;1,2-dichloroethane; methylcyclohexane; N-methylpyrrolidone;nitromethane; acetone; DMSO; ethyl acetate; ethyl ether; and ethylformate. To this solution was added triphosgene (0.4 to 5 eq.), and themixture was stirred at 23° C. until complete reaction as determined byHPLC (<5% 1i or 1j remaining) or TLC. The reaction mixture was washedwith saturated bicarbonate (enough to neutralize any generated HCl), theorganic layer dried with sodium or magnesium sulfate (until no clumpsform), filtered, concentrated, and dried in vacuo at not more than 50°C. until loss on drying (LOD) was less than 0.1%. The dried intermediatewas suspended in a mixture of anhydrous methanol (˜21 liters per mole ofintermediate used) and anhydrous ammonia gas (˜22 eq.), and the mixturewas stirred for not less than 48 hours and until complete consumption ofintermediate product. After reaction completion excess ammonia wasremoved by application of a slight vacuum for one hour before themixture was concentrated in vacuo at 50±5° C. to a minimal volume (˜6liters per mole of intermediate used) and cool to 5±5° C. initiatecrystallization of the crude product. The crude product was filteredafter 1 hour, washed with cold (5° C.) methanol (0.5 liter per mole ofintermediate used), dissolved in hot (42° C.) anhydrous methanol (33liters per mole of intermediate used), decolorized with activated carbon(equivalent to 24 g per mole of intermediate used), filtered to obtain aclear solution of product, concentrated in vacuo at not more than 50° C.to a minimal volume (˜2 liters per mole of intermediate used), andallowed to crystallize at 22±5° C. for ˜12 hours. The slurry wasfiltered, the crystalline product washed with methanol (0.2 liter permole of intermediate used), and the pure product 12 or 13 dried in vacuoat not more than 40° C. for ˜8 hours.

3. Hydrogen and Halogen Derivatives of Decitabine or Azacitidine

In this example, one or more of the 1′-5′ position in the ribose ring ofdecitabine or azacitidine (R₁ ^(′)-R₆ ^(′), FIG. 6A) are replaced with ahalogen (H, F, Cl, Br, I, or CF₃)

The inventors believe that introducing halogen into decitabine shouldprevent hydrolytic and oxidative cleavage from occurring. Whenintroduced onto the sugar ring, the electronegativity of thefield-effect electron-withdrawing (−I) halogens lowers the surroundingelectron density and prevents oxidative cleavage of the sugar moiety.For this reason, halogen derivatives, especially fluorine, are highlydesirable. One additional benefit of fluorine derivatives is that thefluorine atom is so small that there is no distinct stereoscopicdifference between the C—H bond and the C—F bond, which allows fluorinederivatives to mimic natural C—H bond and be easily incorporated intothe metabolic pathway of living organisms. FIG. 6A illustrates thepositions where hydrogen or halogen substitution can be incorporated.FIGS. 6B and 6C list a series of halogenated derivatives that will bemade, where X is preferably H, F, Cl, Br, I or CF₃.

Halogen such as F, Cl, Br and I are not only field-effectelectron-withdrawing (−I) groups but also resonance-effectelectron-donating (+M) groups. In a conjugated system like the triazinering, the resonance-effect of halogens may stabilize the 6-position andprevent oxidative and hydrolytic cleavage of the ring (FIG. 6D).

The halogen derivatives of decitabine can be synthesized by usinghalogenated pentose and triazine precursors. These precursors can besynthesized by using pre-existing or modified methods known in the art.For example, the precursor1-O-acetyl-2-deoxy-3,5-di-O-benzoyl-2,2-difluoro-D-ribose 1k can beobtained from inexpensive D-glucose and D-mannose (FIG. 7A). Fernández,R.; Matheu, M. I.; Echarri, R.; Castillón, S. Tetrahedron 1998, 54,3523-3532. Other mono-fluorinated ribose derivatives can be obtainedfrom suitably protected ribose. Dax, K.; Albert, M.; Ortner, J.; Paul,B. J. Carbohydr. Res. 2000, 327, 47-86. Pankiewiz, K. W. Carbohydr. Res.2000, 327, 87-105.

In one embodiment, for example, a 1.0:1.5:3.0 molar mixture of1-O-acetyl-2-deoxy-3,5-di-O-benzoyl-2,2-difluoro-D-ribose (1k);2-[(trimethylsilyl)amino]-4-[(trimethylsilyl)oxy]-s-triazine (1n); SnCl₄in anhydrous mixture of acetonitrile-1,2-dichloroethane (3.0:1.0, v/v,at a concentration of 50 mM 1k) was refluxed for not less than 3 hoursor until complete reaction as determined by HPLC (<5% 1k remaining) orTLC. The reaction mixture was diluted with 1,2-dichloroethane, washedwith cold saturated bicarbonate, the organic layer dried with sodium ormagnesium sulfate (until no clumps form), filtered, concentrated, anddried in vacuo at not more than 50° C. until loss on drying (LOD) wasless than 0.1 %. The dried intermediate was suspended in a mixture ofanhydrous methanol (˜21 liters per mole of intermediate used) andanhydrous ammonia gas (˜22 eq.), and the mixture was stirred for notless than 48 hours and until complete consumption of intermediateproduct. After reaction completion excess ammonia was removed byapplication of a slight vacuum for one hour before the mixture wasconcentrated in vacuo at 50±5° C. to a minimal volume (˜6 liters permole of intermediate used) and cool to 5±5° C. initiate crystallizationof the crude product. The crude product was filtered after 1 hour,washed with cold (5° C.) methanol (0.5 liter per mole of intermediateused), dissolved in hot (42 C) anhydrous methanol (33 liters per mole ofintermediate used), decolorized with activated carbon (equivalent to 24g per mole of intermediate used), filtered to obtain a clear solution ofproduct, concentrated in vacuo at not more than 50° C. to a minimalvolume (˜2 liters per mole of intermediate used), and allowed tocrystallize at 22±5° C. for ˜12 hours. The slurry was filtered, thecrystalline product washed with methanol (0.2 liter per mole ofintermediate used), and the pure product 16 (where X═F) dried in vacuoat not more than 40° C. for ˜8 hours.

Azacytidine derivatives 14 and 15 (FIG. 6B) can likewise be synthesizedby coupling similarly protected fluoro-ribose derivatives such as 1l and1m (FIG. 7B) with2-[(trimethylsilyl)amino]-4-[(trimethylsilyl)oxy]-s-triazine (1n) in thepresence of SnCl₄ in acetonitrile-1,2-dichloroethane.

In some embodiment, other Lewis acids (which include, but are notlimited to: TMSOTf, BX₃, AlX₃, FeX₃, GaX₃, SbX₅, SnX₄, AsX₅, ZnX₂, andHgX₂, where X is a halogen) in the range of 0.1 to 3 molar equivalentscan be used to facilitate coupling between protected fluoro-ribosederivatives and2-[(trimethylsilyl)amino]-4-[(trimethylsilyl)oxy]-s-triazine (1n).

Halogenation of the triazine moiety can be performed by modifyingpre-existing procedures. One such procedure is reaction of anodicallygenerated “halonium (X)” ions with protected decitabine or azacitidine(FIG. 7C) Palminsano, G.; Danieli, B; Santagostino, M.; Vodopivec, B.Tetrahedron Lett. 1993, 33, 7779-7782. Another is to treat protecteddecitabine or azacitidine with strong bases such as lithium and sodiumhydride or alkyl lithium such as butyl tert-butyl lithium to generate ananion to react with electrophilic halogenating agents such as(CF₃SO₂)₂NF and Selectfluor™ (Barnette, W. E. J. Am. Chem. Soc. 1984,106, 452-454.), Br₂ (Schwartz, E. B.; Knobler, C. B.; Cram, D. J. J. Am.Chem. Soc. 1992, 114, 10775-10784), and I₂ (Tsang, Y. K.; Diaz, H.;Graciani, N.; Kelly, J. W. J. Am. Chem. Soc., 1994, 116, 3988-4005) toform halogen-substitution at the 6-position on the triazine ring. Fromthe 6-iodo derivative, interconversion to into 6-fluoro (Lee, S. H.;Swartz, J. J. Am. Chem. Soc.; 1986, 108, 2445-2447), chloro and bromo(Commercon, A.; Normant, J.; Villieras, J. J. Organometallic Chem. 1975,93, 415-421) derivatives is possible.

In one embodiment, for example, azacytidine derivative 44 was preparedby stirring and refluxing (˜90° C.) a mixture of1′-(2′-Deoxy-3′,5′-di-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiruet(1i) in formic acid (1.5 eq.) and 50 equivalents of trimethylorthoformate until complete reaction (<5% 1i remaining) as determined byHPLC. The reaction was cooled to 22±5° C. before charging slowly (in aperiod of not less than 2 hours) with water (not less than 3 kg of waterper mole of 1i used) to precipitate the fully protected intermediate(FIG. 7D). The mixture was stirred for an additional one hour before itwas cooled to 5±5° C. and stirred for two hours. The slurry was filteredand the solid cake was dried in vacuo at 50±5° C. until loss on drying(LOD) was less than 0.1%. The protected intermediate (1o) was dissolved(0.001 to 10 M concentration) in an anhydrous aprotic organic solventeffective in solubilizing it. These solvents include, but are notlimited to: acetonitrile; chlorobenzene; dichloromethane;1,2-dichloroethane; methylcyclohexane; N-methylpyrrolidone;nitromethane; acetone; DMSO; ethyl acetate; ethyl ether; and ethylformate. While the reaction mixture was submerged in a bath (−78 to 20°C.), a slight excess of tert-butyllithium solution in pentane was slowlyadded (a period of not less than 30 minutes) before an electrophilicfluorinating agent such as Selectfluor™ (1.1 to 10 eq.) was added. Thereaction mixture was stirred until complete reaction as determined byHPLC (<5% 1o remaining) or TLC. The reaction mixture was diluted withthe solvent, washed with cold saturated ammonium chloride, the organiclayer dried with sodium or magnesium sulfate (until no clumps form),filtered, concentrated, and dried in vacuo at not more than 50° C. untilloss on drying (LOD) was less than 0.1%. The dried intermediate wassuspended in a mixture of anhydrous methanol (˜21 liters per mole ofintermediate used) and anhydrous ammonia gas (˜22 eq.), and the mixturewas stirred for not less than 48 hours and until complete consumption ofintermediate product. After reaction completion excess ammonia wasremoved by application of a slight vacuum for one hour before themixture was concentrated in vacuo at 50±5° C. to a minimal volume (˜6liters per mole of intermediate used) and cooled to 5±5° C. initiatecrystallization of the crude product. The crude product was filteredafter 1 hour, washed with cold (5° C.) methanol (0.5 liter per mole ofintermediate used), dissolved in hot (42° C.) anhydrous methanol (33liters per mole of intermediate used), decolorized with activated carbon(equivalent to 24 g per mole of intermediate used), filtered to obtain aclear solution of product, concentrated in vacuo at not more than 50° C.to a minimal volume (˜2 liters per mole of intermediate used), andallowed to crystallize at 22±5° C. for ˜12 hours. The slurry wasfiltered, the crystalline product washed with methanol (0.2 liter permole of intermediate used), and the pure product 44 (FIG. 7D, whereR_(d)═F and X═H) dried in vacuo at not more than 40° C. for 8 hours.

Azacitidine derivative 44 (FIG. 7D, where R_(d)═F and X═OH) can likewisebe synthesized starting from1′-(2′,3′,′5′-tri-O-p-chlorobenzoyl-D-ribofuranosyl)-4-O-methylisobiruet(1i).

The sequence of incorporation of the functional groups can be determinedby one of ordinary skill in the art based on the yield and efficiency ofthe reactions.

4. Alkyl-Halogen Derivatives of Decitabine or Azacitidine

FIGS. 8A, B, D, E, F, G list preferred examples of decitabine andazacitidine derivatives with combined modifications at the 4- or6-position and at the 2′, 3′ and/or 5′ position. The inventors believethat combining modifications on the triazine and ribose ring shouldstabilize the derivatives and render them more resistant to hydrolyticcleavages than those with modification on the triazine or ribose ringalone.

The procedure used to synthesize 1i and 1j can be adapted to prepare ananalogous fluoro-intermediate 1q (FIG. 8C), from which subsequent fluoroderivative 46 can be synthesized. Likewise, other fluoro-intermediatesare accessible.

In one embodiment, for example, azacytidine derivative 46 was preparedby stirring and refluxing (˜90° C.) a mixture of1′-(2′-deoxy-3′,5′-di-O-p-chlorobenzoyl-2′,2′-difluoro-D-ribofuranosyl)-4-O-methylisobiruet(1q) in formic acid (1.5 eq.) and 50 equivalents of trimethylorthoformate until complete reaction (<5% 1q remaining) as determined byHPLC. The reaction was cooled to 22±5° C. before charging slowly (in aperiod of not less than 2 hours) with water (not less than 3 kg of waterper mole of 1q used) to precipitate the fully protected intermediate(1r, FIG. 8C). The mixture was stirred for an additional one hour beforeit was cooled to 5±5° C. and stirred for two hours. The slurry wasfiltered and the solid cake was dried in vacuo at 50±5° C. until loss ondrying (LOD) was less than 0.1%. The protected intermediate (1r) wasdissolved (0.001 to 10 M concentration) in an anhydrous aprotic organicsolvent effective in solubilizing it. These solvents include, but arenot limited to: acetonitrile; chlorobenzene; dichloromethane;1,2-dichloroethane; methylcyclohexane; N-methylpyrrolidone;nitromethane; acetone; DMSO; ethyl acetate; ethyl ether; and ethylformate. While the reaction mixture was submerged in a bath (−78 to 20°C.), a slight excess of tert-butyllithium solution in pentane was slowlyadded (a period of not less than 30 minutes) before an electrophilicfluorinating agent such as Selectfluor™ (1.1 to 10 eq.) was added. Thereaction mixture was stirred until complete reaction as determined byHPLC (<5% 1r remaining) or TLC. The reaction mixture was diluted withthe solvent, washed with cold saturated ammonium chloride, the organiclayer dried with sodium or magnesium sulfate (until no clumps form),filtered, concentrated, and dried in vacuo at not more than 50° C. untilloss on drying (LOD) was less than 0.1%. The dried intermediate wassuspended in a mixture of anhydrous methanol (˜21 liters per mole ofintermediate used) and anhydrous ammonia gas (˜22 eq.), and the mixturewas stirred for not less than 48 hours and until complete consumption ofintermediate product. After reaction completion excess ammonia wasremoved by application of a slight vacuum for one hour before themixture was concentrated in vacuo at 50±5° C. to a minimal volume (˜6liters per mole of intermediate used) and cooled to 5±5° C. to initiatecrystallization of the crude product. The crude product was filteredafter 1 hour, washed with cold (5° C.) methanol (0.5 liter per mole ofintermediate used), dissolved in hot (42° C.) anhydrous methanol (33liters per mole of intermediate used), decolorized with activated carbon(equivalent to 24 g per mole of intermediate used), filtered to obtain aclear solution of product, concentrated in vacuo at not more than 50° C.to a minimal volume (˜2 liters per mole of intermediate used), andallowed to crystallize at 22±5° C. for ˜12 hours. The slurry wasfiltered, the crystalline product washed with methanol (0.2 liter permole of intermediate used), and the pure product 46 (FIG. 8C, whereR_(d)═H and X═F) dried in vacuo at not more than 40° C. for 8 hours.

The procedure used to synthesize 1i and 1j can be adapted to prepare ananalogous fluoro-intermediate is (FIG. 8H), from which subsequent fluoroderivatives it and 75 can be synthesized. Likewise, otherfluoro-intermediates are accessible.

5. Synthesis of Decitabine Analog 1′d

1) Synthesis of 1′d-iii:

Silver cyanate (0.925 g, 6.05 mmol) was co-distilled with dry toluene(2×25 mL) which was then stirred in dry toluene (40 mL) under nitrogenatmosphere (FIG. 9). The reaction temperature was then raised to 55° C.and compound C-137 (2 g, 2.32 mmol) was added to it at 55° C. Thereaction was maintained at 50-55° C. for 2 h. After completion of thereaction, as monitored by TLC (10% ethyl acetate/toluene; the reactionsample was prepared by diluting the clear solution with an equal volumeof ethanol and allowing to stand for 15 min before spotting; C-137 wasalso dissolved in ethanol), the mixture was filtered through celite andthe filtrate (1′d-i, 2 g) was used for next step as such.

Compound 1′d-i (2 g, 4.75 mmol) in toluene was cooled to 15° C. andstirred at the same temperature for 15 min. N-Methylguanidinehydrochloride (1′d-ii, 1.96 g, 4.58 mmol) was treated with 20% aqueouspotassium hydroxide solution to pH=9 and the resultant free base wasadded to 1′d-i at 15° C. The mixture was then stirred at 15° C. for 10min and at RT overnight. After completion of the reaction, as monitoredby TLC, the reaction was filtered to separate the solid formed in thereaction. The off-white solid (1′d-iii) was washed with hexane and thetoluene layer from the filtrate was separated, concentrated and theobtained solid was mixed with earlier separated 1′d-iii (2.0 g, 90.90%).Mass: 508.8 [M+H]⁺.

2) Synthesis of 1′d-iv:

To 1′d-iii (2 g, mmol) triethyl orthoformate (32 mL, 192.93 mmol) wasadded under nitrogen atmosphere. Formic acid (0.2 mL, 5.90 mmol) wasadded to it and the mixture was maintained at 100° C. for 4 h. Aftercompletion of the reaction, as monitored by TLC, water (50 mL) was addedto it. The mixture was stirred for 10 min at RT and was extracted intoethyl acetate (100 mL). The organic layer was washed with brine andwater (50 mL), dried over Na₂SO₄and concentrated to yield crude 1′d-iv(1.9 g). The crude was purified by column chromatography using ethylacetate/hexane to obtain 400 mg of 1′d-iv (20%). Mass: 518.8 [M+H]⁺.

3) Synthesis of Analog 1′d:

A solution of 1′d-iv (0.44 g, 0.456 mmol) was stirred in methanol (20mL) with 3 Å molecular sieves for 1 h at 10-15° C. under nitrogenatmosphere. Potassium carbonate (0.175 g, 1.27 mmol) was added to themixture in 2 lots under nitrogen atmosphere. The reaction was maintainedat RT for 48 h. After completion of the reaction, as monitored by TLC,the mixture was neutralized with 10% acetic acid/methanol solution. Themixture was filtered on celite and the filtrate was concentrated undervacuum. The crude (200 mg) was purified by silica gel columnchromatography using methanol/DCM to get analog 1′d as a white solid(0.085 g, 42.5%). Mass (C₉H₁₄N₄O₄: exact mass 242.10): found, 243[M+H]⁺, 264.9 [M+Na]⁺, 484.9 [Dimer+H]⁺, 506.9 [Dimer+Na]⁺; ¹HNMR (200MHz, CD₃OD) δ: 8.6 (s, 1H, Ar—H), 6.2 (m, 1H, H-1′), 4.4 (m, 2H), 4.0(m, 1H), 3.8 (m, 2H), 3.6 (d, 1H), 2.95 (s, 3H, NH—CH₃), 2.6-2.8 (m,1H), 2.2-2.55 (m, 3H); HPLC: 93.7030%.

6. Synthesis of Decitabine Analog 1′a

1) Synthesis of 1′a-i:

To D-151 (5.0 g, 9.79 mmol) trimethyl orthoformate (52 mL, 469.92 mmol)was added under nitrogen atmosphere (FIG. 10). Formic acid (0.67 g,14.56 mmol) was added and the reaction was maintained at 90° C. for 10h. After completion of the reaction, as monitored by TLC, water (35 mL)was added to the reaction and the mixture was stirred for 1 h at RT. Thereaction was cooled to 3° C. and maintained at 3-5° C. for another onehour. The solid that separated was filtered and dried under vacuum at60-65° C. to get 1′a-i (3.6 g, 70.79%) as off-white solid. Mass: 519.7[M+H]⁺, 128.0 [Base+H]⁺.

2) Synthesis of Analog 1′a:

Compound 1′a-i (2.4 g, 9.34 mmol) was stirred in methanol (25 mL) with 3Å molecular sieves for 1 h at 10-15° C. under nitrogen atmosphere.Potassium carbonate (0.21 g, 1.52 mmol) was stirred in 25 mL of methanolwith 3 Å molecular sieves for 1 h at RT. The resultant potassiumcarbonate/methanol solution was added to 1′a-i under nitrogenatmosphere. The reaction was maintained at 10-15° C. for 15 min and thenstirred for 3 h at RT. After completion of the reaction, as monitored byTLC, the mixture was neutralized with 10% acetic acid/methanol solution.The mixture was filtered on celite and the filtrate was concentrated invacuo. The residue (2.0 g) was purified by silica gel columnchromatography using DCM/methanol; the column was eluted isocraticallyusing 92.5:7.5 DCM/MeOH. Unknown species [Rf: 0.54, 0.66, 0.71, 0.74 and0.88] which absorb 254 nm but do not contain sugar residues. Theα-anomer of analog 1′a elutes at R_(f)=0.32. The β-isomer then elutes atR_(f)=0.29. Further washes elute the two ring opened degraded productsas minor components [R_(f)=0.19 & 0.23]. The analog 1′a (0.9 g, 87.5%)was obtained as off-white solid. Mass (C₉H₁₃N₃O₅: exact mass 243.091):244.0 [M+H]⁺, 128.0 [Base+H]⁺; ¹HNMR (200 MHz, CD3OD) δ: 9.00 (s, 1H,Ar—H), 6.2 (m, 1H), 4.5-4.37 (m, 2H), 4.00 (m, 1H), 4.00 (s, 3H, OCH₃),2.7-2.5 (m, 1H), 2.4-2.3 (m, 1H), ¹³CNMR (50 MHz, CD₃OD) ppm: 171.711,159.580, 156.472, 92.497, 90.699, 89.667, 88.816, 72.617, 71.370,63.393, 62.127, 56.386, 50.325, 42.366, 41.736; HPLC: 90.384%

7. Synthesis of Decitabine Analog 7a

1) Synthesis of 7a-i:

To D-151 (6.0 g, 11.7 mmol) triethyl orthoacetate (108 mL, 0.588 moles)was added under nitrogen atmosphere (FIG. 11). Formic acid (0.68 mL,17.6 mmol) was added and the reaction was maintained at 90° C. for 10 h.After completion of the reaction, as monitored by TLC, water (150 mL)was added to it and the mixture was stirred for 10 min at RT. Thereaction was extracted into ethyl acetate (250 mL) and the organic layerwas washed with water (150 mL), dried over Na₂SO₄ and concentrated. Thecrude (5 g) was purified by silica gel column chromatography using ethylacetate/hexane to obtain 7a-i (3 g, 47.7%).

2) Synthesis of Analog 7a:

Compound 7a-i (1.25 g, 2.34 mmol) was stirred in methanol (25 mL) with 3Å molecular sieves for 1 h at 10-15° C. under nitrogen atmosphere.Potassium carbonate (0.106 g, 0.77 mmol) was stirred in 25 mL ofmethanol with 3 Å molecular sieves for 1 h at RT. The resultantpotassium carbonate/methanol solution was added to 7a-i under nitrogenatmosphere. The reaction was maintained at 10-15° C. for 15 min and thenstirred for 12 h at RT. After completion of the reaction, as monitoredby TLC, the mixture was neutralized with 10% acetic acid/methanolsolution. The reaction was filtered on celite and the filtrate wasconcentrated in vacuo. The crude (1.0 g) was purified by silica gelcolumn chromatography using methanol/DCM to yield analog 7a (50 mg,8.3%) as off-white colored solid. Mass (C₉H₁₅N₃O₅: exact mass 257.10):537.2 [Dimer+Na]⁺, 280.1 [M+Na]⁺, 142.1 [Base+H]⁺. ¹HNMR (200 MHz,CD₃OD) δ: 6.4-6.2 (m, 1H), 4.6-4.5 and 4.4-4.3 (m, 1H), 4.0 (s, 3H,OCH₃), 4.0-3.6 (m, 3H), 2.7 (s, 3H, Ar—CH₃), 3.0-2.8 (m, 1H), 2.-4-2.2(m, 1H). HPLC: 17.63+75.03% (92.6%).

8. Synthesis of Decitabine Analog 134

1) Synthesis of 134-iii:

Silver cyanate (2.1 g, 14.01 mmol) was co-distilled with dry toluene(2×25 mL) and then stirred in dry toluene (100 mL) under nitrogenatmosphere (FIG. 12). The reaction temperature was then raised to 55° C.and Compound C-137 (5 g, 11.68 mmol) was added to it at 55° C. Thereaction was maintained at 50-55° C. for 2 h. After completion of thereaction, as monitored by TLC (10% ethyl acetate/toluene; the reactionsample was prepared by diluting the clear solution with an equal volumeof ethanol and allowing to stand for 15 min before spotting; C-137 wasalso dissolved in ethanol), the mixture was filtered through celite andfiltrate (134-i, 5 g) was used as such for the next step.

Intermediate 134-i (5 g, 11.48 mmol) was stirred at 15° C. for 15 min.Reagent 2-methyl-2-thiopseudourea sulfate (134-ii, 6.17 g, 44.38 mmol)was treated with 20% potassium hydroxide solution to pH=9. The resultantfree base was added to 134-ii at 15° C., the mixture was stirred at 15°C. for 10 min and at RT overnight. After completion of the reaction, asmonitored by TLC, the solid that separated in the reaction was filteredand washed with hexane to get 134-iii (3.7 g, 60.6%) as a white solid.Mass: 525.9 [M+H]⁺; ¹HNMR (200 MHz, DMSO-d₆) δ: 8.1-7.95 (m 2H, Ar—H),7.75-706 (m, 2H, Ar—H), 7.2 (m, 1H, H-1′), 5.9-5.7 (m, 1H), 5.5-5.3 (m,1H), 4.5 (m, 2H), 2.35 (s, 3H, S—CH₃), 2.8-2.6 (m, 1H), 2.6-2.4 (m, 1H).

2) Synthesis of 134-iv:

To 134-iii (2 g, 3.8 mmol) trimethyl orthoformate (21.1 mL, 190.4 mmol)was added under nitrogen atmosphere. Then formic acid (0.22 mL, 5.7mmol) was added and the reaction was maintained at 90° C. for 10 h.After completion of the reaction, as monitored by TLC, water (15 mL) wasadded to it and the mixture was stirred for 1 h at RT. The reaction wasthen cooled to 3° C. and maintained at 3-5° C. for another one hour. Theresultant solid was filtered and dried under vacuum at 60-65° C. toobtain 134-iv (1.15 g, 56.4%) as off-white solid. Mass: 535.7 [M+H]⁺.

3) Synthesis of Analog 134:

Compound 134-iv (1.15 g, 2.14 mmol) was stirred in methanol (25 mL) with3 Å molecular sieves for 1 h at 10-15° C. under nitrogen atmosphere.Potassium carbonate (0.148 g, 1.07 mmol) was stirred in 50 mL ofmethanol with 3 Å molecular sieves for 1 h at RT. The resultantpotassium carbonate/methanol solution was added to 134-iv under nitrogenatmosphere, the reaction was maintained at 10-15° C. for 15 min and thenstirred for 3 h at RT. After completion of the reaction, as monitored byTLC, the mixture was neutralised with 10% Acetic acid/Methanol solution.The mixture was filtered on celite and the filtrate was concentratedunder vacuum. The crude (1.5 g) was purified by column chromatographyusing methanol/DCM to get analog 134 (0.08 g, 14.4%). Mass (C₉H₁₃N₃O₄S:exact mass 259.06): 260.0 [M+H]⁺; ¹HNMR (200 MHz, CH₃OD) δ: 8.95 (s, 1H,Ar—H), 6.2 (m, 1H, H-1′), 5.1 (m, 1H), 4.5 (m, 2H), 2.5 (s, 3H, S—CH₃),2.0-2.3 (m, 1H), ¹³CNMR (50 MHz, CD₃OD) ppm: 155.767, 106.523, 92.533,90.592, 89.718, 88.756, 88.149, 72.769, 72.622, 72.353, 71.391, 64.893,63.375, 63.175, 62.124, 55.377, 50.327, 49.901, 49.475; HPLC:22.44+74.78% (97.22%).

9. Synthesis of Decitabine Analog 7c

1) Synthesis of 7c-i:

To D-151 (6.0 g, 11.7 mmol) triethyl orthoacetate (108 mL, 0.588 moles)was added under nitrogen atmosphere (FIG. 13). Formic acid (0.68 mL,17.6 mmol) was added and the reaction was maintained at 90° C. for 10 h.After completion of the reaction, as monitored by TLC, water (150 mL)was added to it and the mixture was stirred for 10 min at RT. Themixture was extracted into ethyl acetate (250 mL) and the organic layerwas washed with water (150 mL), dried over Na₂SO₄ and concentrated. Thecrude (5 g) was purified by silica gel column chromatography using ethylacetate/hexane to obtain 7c-i (3 g, 47.7%).

2) Synthesis of 7c:

Compound 7c-i (0.5 g, 0.93 mmol) was stirred in methanol (25 mL) with 3Å molecular sieves for 1 h at 10-15° C. under nitrogen atmosphere.1,1-Dimethylammonium chloride (0.115 g, 1.4 mmol) in methanol (5 mL) wastreated with potassium hydroxide (0.080 g, 1.4 mmol) in methanol (10 mL)and stirred for 20 min at RT. The resultant solution was added to 7c-iunder nitrogen atmosphere. The reaction was maintained at 10-15° C. for15 min and then stirred for 12 h at RT. After completion of thereaction, as monitored by TLC, the mixture was neutralised with 10%acetic acid/methanol solution. The reaction was filtered on celite andthe filtrate was concentrated under vacuo at 35-40° C. The crude (0.55g) was purified by silica gel column chromatography using methanol/DCMto yield 7c (80 mg, 32%). Mass: 155.0 [Base+H]⁺, 270.9 [M+H]⁺, 292.9[M+Na]⁺, 540.7 [Dimer+H]⁺, 562.7 [Dimer+Na]⁺; ¹HNMR (200 MHz, CDCl₃) δ:6.0 (m, 1H), 3.9-3.7 (m, 2H), 3.3-3.2 (2s, 6H, N—(CH₃)₂), 2.6 (s, 3H,CH₃); HPLC: 97.69%.

10. Synthesis of Decitabine Analog 135

1) Synthesis of 135-iii:

Silver thiocyanate (0.5 g, 3.0 mmol) was co-distilled with dry toluene(2×25 mL) and then stirred in dry toluene (40 mL) under nitrogenatmosphere (FIG. 14. The temperature was raised to 55° C. and compoundC-137 (1 g, 2.3 mmol) was added to it at 55° C. The reaction wasmaintained at 50-55° C. for 2 h. After completion of the reaction, asmonitored by TLC (10% ethyl acetate/toluene; the reaction sample wasprepared by diluting the clear solution with an equal volume of ethanoland allowing to stand for 15 min before spotting; C-137 was alsodissolved in ethanol), the mixture was filtered through celite and thefiltrate (135-i, 1 g) was used as such for the next step.

Intermediate 135-i (1 g, 2.28 mmol) in toluene was cooled to 15° C. andmaintained for 15 min. N-Methylguanidine hydrochloride (135-ii, 0.925 g,8.4 mmol) was treated with 20% aqueous potassium hydroxide solution topH=9 and the resultant free base was added to 135-ii at 15° C. Themixture was stirred at 15° C. for 10 min and at RT overnight. Aftercompletion of the reaction, as monitored by TLC, the solid thatseparated in the reaction was filtered, washed with hexane and dried toget 135-iii (0.7 g, 60%) as off-white solid. Mass: 524.8 [M+H]⁺, 562.7[M+Na]⁺.

2) Synthesis of 135-iv:

To compound 135-iii (0.55 g, 1.04 mmol) trimethyl orthoformate (8.72 mL,52 mmol) was added under nitrogen atmosphere. Formic acid (0.06 mL, 1.55mmol) was added to it and the mixture was maintained at 90-95° C. for 7h. After completion of the reaction, as monitored by TLC, water (50 mL)was added to it. The mixture was stirred for 10 min at RT and wasextracted in Ethyl acetate (100 mL). The organic layer was washed withwater (50 mL), dried over sodium sulfate and concentrated. The crude(400 mg) was purified by silica gel column chromatography using ethylacetate/hexane to obtain 215 mg (39%) of 135-iv. Mass: 534.7 [M+H]⁺.

3) Synthesis of Analog 135:

Compound 135-iv (0.20 g, 0.374 mmol) was stirred in methanol (20 mL)with 3 Å molecular sieves for 1 h at 10-15° C. under nitrogenatmosphere. Potassium carbonate (0.025g, 0.18 mmol) was stirred in 20 mLof methanol with 3 Å molecular sieves for 1 h at RT. The resultantpotassium carbonate/Methanol solution was added to 135-iv under nitrogenatmosphere and the mixture was maintained at 10-15° C. for 15 min andfor 12 h at RT. After completion of the reaction, as monitored by TLC,the mixture was neutralized with 10% acetic acid/methanol solution. Thereaction was filtered on celite and the filtrate was concentrated undervacuo. The crude (200 mg) was purified by silica gel columnchromatography using methanol/DCM to yield analog 135 (0.025 g, 25.5%)as white colored solid. Mass (C₉H₁₄N₄O₃S: exact mass 258.08): 259.0[M+H]⁺, 281.0 [M+Na]⁺; ¹HNMR (CD₃OD, 200 MHz) δ: 8.3 (s, 1H, Ar—H), 6.7and 6.85 92m, 1H, H-1′), 4.4-4.6 (m, 2H), 3.8-40 (m, 2H), 3.6 (d, 2H),3.0 (s, 3H, NH—CH₃), 2.6-2.8 (m, 2H); HPLC: 59.73+34.09% (93.82%).

11. Synthesis of Decitabine Analog 136

1) Synthesis of 136-iii:

Silver thiocyanate (0.925 g, 5.6 mmol) was co-distilled with dry toluene(2×25 mL) and then stirred in dry toluene (60 mL) under nitrogenatmosphere (FIG. 15). The temperature of the mixture was raised to 55°C. and compound C-137 (2 g, 4.67 mmol) was added to it at 55° C. Thereaction was maintained at 50-55° C. for 2 h. After completion of thereaction, as monitored by TLC (10% ethyl acetate/toluene; the reactionsample was prepared by diluting the clear solution with an equal volumeof ethanol and allowing to stand for 15 min before spotting; C-137 wasalso dissolved in ethanol), the mixture was filtered through celite andthe filtrate (136-i, 2 g) was used for next step as such.

Intermediate 136-i (2 g, 4.57 mmol) in toluene was cooled to 15° C. andstirred at the same temperature for 15 min. 1,1-Dimethylguanidinesulfate (136-ii, 4.6 g, 16.8 mmol) was treated with 20% aqueouspotassium hydroxide solution to pH=9. The resultant free base was addedto 136-i in toluene at 15° C. The mixture was stirred at 15° C. for 10min and at RT overnight. After completion of the reaction, as monitoredby TLC, the solid that separated in the reaction was filtered and washedwith hexane. 136-iii (0.55 g, 22%) was obtained as off-white solid.Mass: 538.9 [M+H]⁺.

2) Synthesis of 136-iv:

To 136-iii (0.55 g, 1.03 mmol) trimethyl orthoformate (7.67 g, 51.76mmol) was added under nitrogen atmosphere. Formic acid (71 mg, 1.55mmol) was added and the reaction was maintained at 90° C. for 10 h.After completion of the reaction, as monitored by TLC, water (50 mL) wasadded and the mixture was stirred for 10 min at RT. The reaction wasextracted in ethyl acetate (100 mL) and the organic layer was washedwith water (50 mL), dried over sodium sulfate and concentrated to yieldcrude 136-iv (300 mg). The crude was purified by silica gel columnchromatography to obtain 250 mg (44.56%) of 136-iv. Mass: 548.8 [M+H]⁺.

3) Synthesis of Analog 136:

Compound 136-iv (0.25 g, 0.456 mmol) was stirred in methanol,(20 mL)with 3 Å molecular sieves for 1 h at 10-15° C. under nitrogenatmosphere. Potassium carbonate (0.062 g, 0.15 mmol) with 3 Å molecularsieves was stirred for 1 h in 20 mL of methanol at RT. The resultantpotassium carbonate/methanol solution was added to 136-iv under nitrogenatmosphere and the reaction was maintained at 10-15° C. for 15 min andthen stirred for 12 h at RT. After completion of the reaction, asmonitored by TLC, the mixture was neutralised with 10% aceticacid/methanol solution. The mixture was filtered on celite and thefiltrate was concentrated in vacuo. The residue (200 mg) was purified bysilica gel column chromatography to yield analog 136 (0.08 g, 64.51%) aswhite colored solid. Mass (C₁₀H₁₆N₄O₃S: exact mass 272.09): 273.0[M+H]⁺; ¹HNMR (CD₃OD, 200 MHz) δ: 8.8 and 8.4 (2s, 1H, Ar—H), 6.85 and6.8 (d and t, 1H, H-1′), 4.4-4.6 (m 2H), 3.8-4.2 (m, 2H), 3.75 (d, 1H),3.35 (2s, 6H, N—(CH₃)₂), 2.8-2.6 (m, 2H), ¹³CNMR (50 MHz, CD₃OD):154.714, 154.674, 93.613, 92.762, 91.322, 89.403, 72.587, 71.310,63.529, 62.057, 42.828, 42.721; HPLC: 46.20+52.97% (99.17%).

12. Synthesis of Decitabine Analog 137

1) Synthesis of 137-iii:

Silver thiocyanate (5 g, 30.12 mmol) was co-distilled with dry toluene(2×125 mL) and stirred in dry toluene (100 mL) under nitrogen atmosphere(FIG. 16). The temperature was raised to 55° C. and compound C-137 (10g, 23.27 mmol) was added to it at 55° C. The reaction was maintained at50-55° C. for 2 h. After completion of the reaction, as monitored by TLC(10% ethyl acetate/toluene; the reaction sample was prepared by dilutingthe clear solution with an equal volume of ethanol and allowing to standfor 15 min before spotting; C-137 was also dissolved in ethanol), themixture was filtered through celite and the filtrate (137-i, 10 g) wasused as such for the next step.

Intermediate 137-i (10 g,) was cooled to 15° C. and stirred at 15° C.for 15 min. O-Methylisourea hydrochloride (14.5 g, 84.22 mmol) wastreated with 20% aqueous potassium hydroxide solution to pH=9. Theresultant free base was added to 137-i at 15° C. The reaction wasstirred at 15° C. for 10 min and at RT overnight. After completion ofthe reaction, as monitored by TLC, the solid that separated in thereaction was filtered and washed with hexane to give 137-iii (7 g, 60%)as white solid. Mass: 525.8 [M+H]⁺.

2) Synthesis of 137-iv:

To 137-iii (10 g, 19.04 mmol) triethyl orthoacetate (175 mL, 933 mmol)was added under nitrogen atmosphere. Formic acid (1.1 mL, 2.8 mmol) wasadded and the reaction was maintained at 90-95° C. for 7 h. Aftercompletion of the reaction, as monitored by TLC, water (150 mL) wasadded to it and the mixture was stirred for 20 min at RT. The reactionwas extracted into ethyl acetate (300 mL) and the organic layer waswashed with water (150 mL), dried over Na₂SO₄ and concentrated. Thecrude (10 g) was purified by silica gel column chromatography usingethyl acetate/hexane to obtain 137-iv (1.6 g, 44.56%). Mass: 551.7[M+H]⁺.

3) Synthesis of 137:

Compound 137-iv (0.3 g, 0.56 mmol) was stirred in methanol (20 mL) with3 Å molecular sieves for 1 h at 10-15° C. under nitrogen atmosphere.Potassium carbonate (0.025 g, 0.18 mmol) was added to 137-iv undernitrogen atmosphere. The reaction was maintained at 10-15° C. for 15 minand then for 1 h at RT. After completion of the reaction, as monitoredby TLC, it was neutralised with 10% acetic acid/methanol solution. Themixture was filtered on celite and the filtrate was concentrated undervacuo. The crude (250 mg) was purified by silica gel columnchromatography using methanol/DCM to yield 137 (0.07 g, 46%) ascolorless thick syrup. Mass: 296.1 [M+Na]⁺, 274.0 [M+H]⁺; ¹HNMR (200MHz, CD₃OD) δ: 6.8-6.6 (m, 1H), 4.4-4.2 (m<1H), 3.8-3.6 (m, 2H), 2.9-2.7(m, 1H), 2.5 (s, 3H, S—CH₃), 2.2-2.0 (m, 1H); HPLC: 90.94%.

13. Inhibition of DNA Methylation by Decitabine Analogs and Derivatives

The demethylating activity of several decitabine analogs and derivativeswere tested in a cell-based GFP (green fluorescent protein) assay. Thisassay involves the use of an expression vector containing a GFP generegulated by the CMV promoter and sensitive to the methylation of CpGsites within the promoter. A decrease in methylation resulting fromexposure to a methylation inhibitor leads to GFP expression and isreadily scored. Specifically, CMV-EE210 cell line containing theepigenetically silenced GFP transgene was used to assay for reactivationof GFP expression by flow cytometry. CMV-EE210 was made by transfectingNIH 3T3 cells with the pTR-UF/UF1/UF2 plasmid (Zolotuhin et al., 1996),which is comprised of pBS(+) (Stratagene, Inc.) containing acytomegalovirus (CMV) promoter driving a humanized GFP gene adapted forexpression in mammalian cells. After transfection, high-level GFPexpressing cells were initially selected by FACS analysis and sortingusing a MoFlo cytometer (Cytomation, Inc.). Decitabine (1a), a potentinhibitor of mammalian DNMT1, was used as a positive control. To screenfor reactivation of CMV-EE210, decitabine or a test compound was addedto complete medium (phenol red free DMEM (Gibco, Life Technologies)supplemented with 10% fetal bovine serum (Hyclone)). Cells were thenseeded to 30% confluence (˜5000 cell/well) in a 96 well plate containingthe test compounds and grown for three days in at 37° C. in 5% CO₂. Theplates were examined under a fluorescent microscope using a 450-490excitation filter (I3 filter cube, Leica, Deerfield Ill.). Wells werescored g1 positive if (1-10%) of viable cells express GFP, g2 positiveif 10-50% of viable cells express GFP, and g3 if 50-100% of the viablecells express GFP. Table 1 summarizes the results of the test fordecitabine, compound 1′a, and compound 7c as DNA methylation inhibitors.As shown in Table 1, both compound 1′a and 7c were able to inhibit DNAmethylation effectively at low concentrations as well, resulting inreactivation of the transcription of the GFP gene. It was also observedthat although decitabine inhibited DNA methylation at a lowerconcentration, it caused extensive necrosis of the cells in the culture.TABLE 1 Demethylation Concentration Compound # (μM) GFP Expression 1a0.6 g3 1'a 2.5 g2 7c 2.5 g114. Stability of Decitabine Analogs and Derivatives in Aqueous Solutionand in the Presence of Cytidine Deaminase (CDA)

The stability of various decitabine analogs and derivatives shown inFIG. 18 was tested in aqueous solution at various pH and in the presenceof CDA. As shown in FIG. 17, at acidic or basic pH's,5-aza-2′-deoxycytidine (decitabine, 1a) degrades rapidly, especially athigher pH's, where the rate of degradation increases exponentiallytoward higher pH's; at neutral pH's, this degradation is minimized.Table 2 summarizes the results of the stability tests for the compoundsshown in FIG. 18. When the 4-NH₂ group of 5-aza-2′-deoxycytidine (1a) isreplaced with NHCH₃ (H vs. CH₃), as illustrated in compound 1′d,stability is greatly increased across pH 5-9. At neutral pH, 83% ofcompound 1′d remains compared to 56% of 5-aza-2′-deoxycytidine (1a)after 24 hr. In addition, compound 1′d is completely resistant todegradation via deamination by CDA (97% vs. 4% after 24 hr).

When the 4-NH₂ group is replaced instead by the 4-SCH₃ group in analog134, stability was greatly improved at pH 5 (91% vs.<50% for 1a after 24hr); stability improvement at pH 7 was modest (72% vs. 56%) to none atpH 9. In addition to being resistant to degradation via CDA deamination,compound 7c was found to be stable across the pH 5-9, which demonstratedthat the destabilizing effect of the 6-CH₃ substitution could becountered with substitution at the 4-position, in this case the4-N(CH₃)₂ group.

When the 4-NH₂ and 2-carbonyl (═O) groups are replaced instead by the4-NHCH₃ and 2-thionyl (═S) groups in compound 135, stability across pH5-9 was slightly lower than compound 1′d; when the 4-position wassubstituted with a 4-N(CH₃)₂ group in compound 136, stability wasrestored to the same levels as compound 1′d. Surprisingly, substitutionof the 2-carbonyl position of analog 7a with 2-thionyl, as embodied inanalog 137, increases stability at neutral and basic pH's (77% vs. 25%at pH 7 and 74% vs. 11% at pH 9 after 24 hr). TABLE 2 % Peak Arearelative to 0 hr pH5* pH 7* pH 9* CDA** Compound # 6 hr 24 hr 6 hr 24 hr6 hr 24 hr 1.5 hr  1a — — 83.94 56.44 — — 4.29  1′d 95.83 84.30 98.5583.76 97.68 91.05 97.03  1′a — — 76.96 62.15 — — —  7a 53.85 24.25 72.9924.83 49.36 11.33 — 134 98.33 90.68 91.66 72.53 73.65 35.01 —  7c 92.9474.20 100.93 100.29 98.90 96.75 100.11 135 70.55 49.49 101.13 93.1689.73 69.17 93.54 136 94.35 76.27 97.54 88.48 95.50 83.42 96.28 13780.19 38.52 94.01 77.06 93.67 73.74 —*The samples were stored at room temperature before analysis.**The reaction mixture contains 0.2 mM substrate in 0.1M Tris.HCl with0.07-0.10 units of recombinant CDA at 37-39° C.

While the present invention is disclosed with reference to preferredembodiments detailed above, it is to be understood that theseembodiments are intended in an illustrative or exemplary rather than ina limiting sense, as it is contemplated that modifications will readilyoccur to those skilled in the art, modifications which will be withinthe spirit of the invention and the scope of the appended claims. Allpatents, papers, articles, references and books cited herein areincorporated by reference in their entirety.

1. A compound of Formula I,

or its pharmaceutically acceptable salt, wherein Rx is hydrogen,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, halogen-substituted alkyl or aryl, phenyl, benzyl,hydroxyl, thiol, substituted or unsubstituted amino, —O-alkyl, —S-alkyl,—O-aryl or —S-aryl; R_(y) is hydrogen, alkyl, or sugar; R_(z) is ahydrogen, alkyl, aryl, arylalkyl, amino, alkylamino, dialkylamino,alkylarylamino, hydrazine, hydroxylalkylamino, —O-alkyl, —S-alkyl,—O-aryl or —S-aryl; and Q is oxygen, sulfur, methylene, imine, oralkylimine; provided that when R_(x) is hydrogen, methyl, chloromethyl,phenyl or benzyl, R_(y) is hydrogen or glycosyl, and Q is oxygen, R_(z)is not amino.
 2. The compound or its pharmaceutically acceptable saltaccording to claim 1, wherein R_(x) is mono-, di- or trifluoromethyl. 3.The compound or its pharmaceutically acceptable salt according to claim1, wherein R_(x) is a straight or branched chain C₁₋₆ alkyl.
 4. Thecompound or its pharmaceutically acceptable salt according to claim 1,wherein R_(x) is selected from the group consisting of methyl, ethyl,propyl, butyl and phenyl.
 5. The compound or its pharmaceuticallyacceptable salt according to claim 1, wherein R_(x) is fluoride.
 6. Thecompound or its pharmaceutically acceptable salt according to claim 1,wherein R_(x) is chloride or bromide.
 7. The compound or itspharmaceutically acceptable salt according to claim 1, wherein R_(x) isprimary or secondary amino.
 8. The compound or its pharmaceuticallyacceptable salt according to claim 1, wherein R_(y) is a substituted orunsubstituted ribose.
 9. The compound or its pharmaceutically acceptablesalt according to claim 1, wherein R_(y) is 2′-deoxyribose or ribose.10. The compound or its pharmaceutically acceptable salt according toclaim 9, wherein R_(x) is halogen, and R_(z) is amino.
 11. The compoundor its pharmaceutically acceptable salt according to claim 9, whereinR_(x) is fluoride, and R_(z) is amino.
 12. The compound or itspharmaceutically acceptable salt according to claim 9, wherein the R_(x)is alkyl, and R_(z) is OCH₃ or O-alkyl.
 13. The compound or itspharmaceutically acceptable salt according to claim 9, wherein the R_(x)is alkyl, R_(y) is 2′-deoxyribose and R_(z) is dimethylamino.
 14. Thecompound or its pharmaceutically acceptable salt according to claim 1,wherein Q is sulfur, and R_(z) is hydrogen, amino, alkylamino,dialkylamino, or O-alkyl.
 15. The compound or its pharmaceuticallyacceptable salt according to claim 14, wherein R_(x) is alkyl, orhalogen.
 16. The compound or its pharmaceutically acceptable saltaccording to claim 1, wherein the salt is selected from the groupconsisting of hydrochloride, mesylate, EDTA, sulfite, L-Aspartate,maleate, phosphate, L-Glutamate, (+)-L-Tartrate, citrate, L-Lactate,succinate, acetate, hexanoate, butyrate, and propionate salt.
 17. Acompound of Formula II

or its pharmaceutically acceptable salt, wherein R_(x) is hydrogen,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, halogen-substituted alkyl or aryl, phenyl, benzyl,hydroxyl, thiol, substituted or unsubstituted amino, —O-alkyl, —S-alkyl,—O-aryl or —S-aryl; R_(z) is a hydrogen, amino, alkyl, aryl, arylalkyl,alkylamino, dialkylamino, alkylarylamino, hydrazine, hydroxylalkylamino,—O-alkyl, —S-alkyl, —O-aryl or —S-aryl; and Q is oxygen, sulfur,methylene, imine, or alkylimine; Q is oxygen, sulfur, methylene, imine,or alkylimine; provided that when R_(x) is hydrogen, methyl,chloromethyl, phenyl, or benzyl, and Q is oxygen, R_(z) is not amino.18. The compound or its pharmaceutically acceptable salt according toclaim 17, wherein the salt is selected from the group consisting ofhydrochloride, mesylate, EDTA, sulfite, L-Aspartate, maleate, phosphate,L-Glutamate, (+)-L-Tartrate, citrate, L-Lactate, succinate, acetate,hexanoate, butyrate, and propionate salt.
 19. A compound of Formula III

or its pharmaceutically acceptable salt, wherein R_(x) is hydrogen,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, halogen-substituted alkyl or aryl, phenyl, benzyl,hydroxyl, thiol, substituted or unsubstituted amino, —O-alkyl, —S-alkyl,—O-aryl or —S-aryl; R_(z) is a hydrogen, amino, alkyl, aryl, arylalkyl,alkylamino, dialkylamino, alkylarylamino, hydrazine, hydroxylalkylamino,—O-alkyl, —S-alkyl, —O-aryl or —S-aryl; and Q is oxygen, sulfur,methylene, imine, or alkylimine; and Q is oxygen, sulfur, methylene,imine, or alkylimine; provided that when R_(x) is hydrogen, methyl,chloromethyl, phenyl, or benzyl, Q is oxygen, R_(z) is not amino. 20.The compound or its pharmaceutically acceptable salt according to claim19, wherein the salt is selected from the group consisting ofhydrochloride, mesylate, EDTA, sulfite, L-Aspartate, maleate, phosphate,L-Glutamate, (+)-L-Tartrate, citrate, L-Lactate, succinate, acetate,hexanoate, butyrate, and propionate salt.
 21. A compound of Formula IV

or its pharmaceutically acceptable salt, wherein R_(x) is hydrogen,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, halogen-substituted alkyl or aryl, phenyl, benzyl,hydroxyl, thiol, substituted or unsubstituted amino, —O-alkyl, —S-alkyl,—O-aryl or —S-aryl; and R_(z) is a hydrogen, amino, alkyl, aryl,arylalkyl, alkylamino, dialkylamino, alkylarylamino, hydrazine,hydroxylalkylamino, —O-alkyl, —S-alkyl, —O-aryl or —S-aryl; each of R₁′,R₂′, R₃′, R₄′, R₅′, and R₆′ is independently selected from the groupconsisting of hydrogen, hydroxyl, fluoride, choloride, bromide, iodide,CF₃, —O-alkyl, —O-acyl, —O-aryl, —S-alkyl, and —S-aryl; and Q is oxygen,sulfur, methylene, imine, or alkylimine, provided that when R_(x) ishydrogen and R_(z) is amino, R₄′ is not hydroxyl.
 22. The compound orits pharmaceutically acceptable salt according to claim 21, wherein R₄′is hydrogen; and each of R₁′, R₂′, R₃′, R₅′, and R₆′ is independentlyselected from the group consisting of hydrogen, hydroxyl, fluoride,chloride, bromide, iodide, CF₃, —O-alkyl, —O-acyl, —O-aryl, —S-alkyl,and —S-aryl.
 23. The compound or its pharmaceutically acceptable saltaccording to claim 21, wherein the salt is selected from the groupconsisting of hydrochloride, mesylate, EDTA, sulfite, L-Aspartate,maleate, phosphate, L-Glutamate, (+)-L-Tartrate, citrate, L-Lactate,succinate, acetate, hexanoate, butyrate, and propionate salt.
 24. Saltof a compound of Formula I,

wherein R_(x) is hydrogen, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted aryl, halogen-substituted alkyl or aryl,phenyl, benzyl, hydroxyl, thiol, substituted or unsubstituted amino,—O-alkyl, —S-alkyl, —O-aryl or —S-aryl; R_(y) is hydrogen, alkyl, orsugar; R_(z) is a hydrogen, amino, alkyl, aryl, arylalkyl, alkylamino,dialkylamino, alkylarylamino, hydrazine, hydroxylalkylamino, —O-alkyl,—S-alkyl, —O-aryl or —S-aryl; and Q is oxygen, sulfur, methylene, imine,or alkylimine, provided that when R_(y) is 2′-deoxy-D-ribose orD-ribose, R_(x) is not hydrogen and R_(z) is not amino.
 25. The salt ofclaim 24, wherein Q is sulfur, and R_(z) is hydrogen, amino, alkylamino,dialkylamino, or O-alkyl.
 26. The salt of claim 25, wherein R_(x) isalkyl, or halogen.
 27. The salt of claim 24, wherein said salt issynthesized with an acid.
 28. The salt of claim 27, wherein said acid isselected from the group consisting of hydrochloric, L-lactic, acetic,phosphoric, (+)-L-tartaric, citric, propionic, butyric, hexanoic,L-aspartic, L-glutamic, succinic, EDTA, maleic, and methanesulfonicacid.
 29. The salt of claim 27, wherein said acid is selected from thegroup consisting of HBr, HF, HI, nitric, nitrous, sulfuric, sulfurous,phosphorous, perchloric, chloric, and chlorous acid.
 30. The salt ofclaim 27, wherein said acid is a carboxylic acid or a sulfonic acid. 31.The salt of claim 30, wherein said carboxylic acid is selected from thegroup consisting of ascorbic, carbonic, and fumaric acid.
 32. The saltof claim 30, wherein said sulfonic acid is selected from the groupconsisting of ethanesulfonic, 2-hydroxyethanesulfonic, andtoluenesulfonic acid.
 33. The salt of claim 24, wherein said salt is ahydrochloride, mesylate, EDTA, sulfite, L-Aspartate, maleate, phosphate,L-Glutamate, (+)-L-Tartrate, citrate, L-Lactate, succinate, acetate,hexanoate, butyrate, or propionate salt.
 34. A pharmaceuticalcomposition comprising a therapeutically-effective amount of an activecompound and a pharmaceutically-acceptable carrier, said active compoundbeing a compound of formula I, II, III, or IV above, or itspharmaceutically acceptable salt.
 35. The pharmaceutical compositionaccording to claim 34, wherein the pharmaceutically-acceptable carrieris an aqueous solution.
 36. The pharmaceutical composition according toclaim 35, wherein the aqueous solution comprises a water misciblenon-aqueous solvent selected from the group consisting of glycerin,propylene glycol, polyethylene glycol, and combinations thereof.
 37. Thepharmaceutical composition according to claim 35, wherein the amount ofthe active compound in the composition is between 0.1 and 200 mg per mlof the aqueous solution.
 38. The pharmaceutical composition according toclaim 34, wherein the pharmaceutical carrier is a solution comprisingless than 40% water and a water miscible non-aqueous solvent.
 39. Thepharmaceutical composition according to claim 38, wherein thenon-aqueous solvent is selected from the group consisting of glycerin,propylene glycol, polyethylene glycol, and combinations thereof.
 40. Thepharmaceutical composition according to claim 34, wherein thepharmaceutical carrier is a solution comprising less than 20% water anda water miscible non-aqueous solvent.
 41. The pharmaceutical compositionaccording to claim 34, being in a form of lyophilized powder.
 42. Thepharmaceutical composition according to claim 34, further comprising anacidifying agent added to the composition in a proportion such that thecomposition has a resulting pH between about 4 and
 8. 43. Thepharmaceutical composition according to claim 42, wherein the acidifyingagent is an inorganic or organic acid.
 44. The pharmaceuticalcomposition according to claim 43, wherein the organic acid is selectedfrom the group consisting of ascorbic acid, citric acid, tartaric acid,lactic acid, oxalic acid, formic acid, benzene sulphonic acid, benzoicacid, maleic acid, glutamic acid, succinic acid, aspartic acid,diatrizoic acid, and acetic acid.
 45. The pharmaceutical compositionaccording to claim 43, wherein the inorganic acid is selected from thegroup consisting of hydrochloric acid, sulphuric acid, phosphoric acid,and nitric acid.
 46. The pharmaceutical composition according to claim43, wherein the acidifying agent is ascorbic acid.
 47. Thepharmaceutical composition according to claim 34, further comprising apharmaceutically acceptable excipient.
 48. The pharmaceuticalcomposition according to claim 47, wherein the excipient is selectedfrom the group consisting of mannitol, sorbitol, lactose, dextrox, andcyclodextrin.
 49. The pharmaceutical composition according to claim 48,wherein the cyclodextrin is α-, β-, or γ-cyclodextrin.
 50. Thepharmaceutical composition according to claim 48, the cyclodextrin is amodified, amorphous cyclodextrin selected from the group consisting ofhydroxypropyl-, hydroxyethyl-, glucosyl-, maltosyl-, maltotriosyl-,carboxyamidomethyl-, carboxymethyl-, sulfobutylether-, anddiethylamino-substituted α-, β- and γ-cyclodextrin.
 51. Thepharmaceutical composition according to claim 34, wherein at least 80%of the active compound remains in active form after storage at 25° C.for 7 days.
 52. The pharmaceutical composition according to claim 34,wherein at least 80% of the active compound remains in active form afterstorage at 25° C. for 14 days.
 53. The pharmaceutical compositionaccording to claim 34, wherein at least 80% of the active compoundremains in active form after storage at 40° C. for 7 days.
 54. Thepharmaceutical composition according to claim 34, further comprising: atherapeutic agent selected from the group consisting of anti-neoplasticagents, alkylating agents, agents that are members of the retinoidssuperfamily, hormonal agents, plant-derived agents, biologic agents,interleukins, interferons, cytokines, immuno-modulating agents, andmonoclonal antibodies.
 55. A kit, comprising: a first vessel containinga compound of formula I, II, III, or IV above, or its pharmaceuticallyacceptable salt in a solid form.
 56. The kit according to claim 55,wherein the active compound is in a form of lyophilized powder.
 57. Thekit according to claim 56, further comprising: a second vesselcontaining a diluent comprising glycerin, propylene glycol, polyethyleneglycol or combinations thereof.
 58. The kit according to claim 57,wherein the diluent comprises less than 40% water in volume.
 59. The kitaccording to claim 55, where the amount of active compound in the firstvessel is between 0.1 and 200 mg.
 60. The kit according to claim 55,wherein the diluent further comprises an acidifying agent.
 61. The kitaccording to claim 55, further comprising: a written instructiondescribing how to administer the active compound as a pharmaceutical toa patient.
 62. A method for treating a patient suffering from a diseaseassociated with aberrant DNA methylation, comprising: administering tothe patient a pharmaceutical composition comprising atherapeutically-effective amount of an active compound and apharmaceutically-acceptable carrier, said active compound being acompound of formula I, II, III, or IV above, or its pharmaceuticallyacceptable salt.
 63. The method according to claim 62, wherein thepharmaceutical composition is administered orally, parenterally,topically, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery, subcutaneously, intraadiposally, intraarticularly,or intrathecally.
 64. The method according to claim 62, wherein thepharmaceutical composition is administered intravenously.
 65. The methodaccording to claim 62, wherein the pharmaceutical composition isadministered subcutaneously.
 66. The method according to claim 62,further comprising: administering to the patient a second therapeuticagent in combination with the pharmaceutical composition.
 67. The methodaccording to claim 66, wherein the second therapeutic agent is selectedfrom the group consisting of antibiotic agents, alkylating agents,retinoids, hormonal agents, plant-derived agents, biologic agents,interleukins, interferons, cytokines, immuno-modulating agents, andmonoclonal antibodies.
 68. The method according to claim 62, wherein thedisease associated with aberrant DNA methylation is selected from thegroup consisting of hematological disorders, benign tumor and cancer.69. The method according to claim 68, wherein the hematological disorderis selected from the group consisting of acute myeloid leukemia, acutepromyelocytic leukemia, acute lymphoblastic leukemia, chronicmyelogenous leukemia, the myelodysplastic syndromes, and sickle cellanemia.